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B Kachar

Publications and source records attributed to B Kachar.

At least 37 records · Page 2Linked to original sources
PubMed2000

Two distinct Ca(2+)-dependent signaling pathways regulate the motor output of cochlear outer hair cells.

The outer hair cells (OHCs) of the cochlea have an electromotility mechanism, based on conformational changes of voltage-sensitive "motor" proteins in the lateral plasma membrane. The translocation of electrical charges across the membrane that accompanies electromotility imparts a voltage dependency to the membrane capacitance. We used capacitance measurements to investigate whether electromotility may be influenced by different manipulations known to affect intracellular Ca(2+) or Ca(2+)-dependent protein phosphorylation. Application of acetylcholine (ACh) to the synaptic pole of isolated OHCs evoked a Ca(2+)-activated apamin-sensitive outward K(+) current. It also enhanced electromotility, probably because of a phosphorylation-dependent decrease of the cell's axial stiffness. However, ACh did not change the voltage-dependent capacitance either in conventional whole-cell experiments or under perforated-patch conditions. The effects produced by the Ca(2+) ionophore ionomycin mimicked those produced by ACh. Hyperpolarizing shifts of the voltage dependence of capacitance and electromotility were induced by okadaic acid, a promoter of protein phosphorylation, whereas trifluoperazine and W-7, antagonists of calmodulin, caused opposite depolarizing shifts. Components of the protein phosphorylation cascade-IP(3) receptors and calmodulin-dependent protein kinase type IV-were immunolocalized to the lateral wall of the OHC. Our results suggest that two different Ca(2+)-dependent pathways may control the OHC motor output. The first pathway modulates cytoskeletal stiffness and can be activated by ACh. The second pathway shifts the voltage sensitivity of the OHC electromotile mechanism and may be activated by the release of Ca(2+) from intracellular stores located in the proximity of the lateral plasma membrane.

Acetylcholine↗
PubMed2000

The motor and tail regions of myosin XV are critical for normal structure and function of auditory and vestibular hair cells.

Recessive mutations in myosin 15, a class XV unconventional myosin, cause profound congenital deafness in humans and both deafness and vestibular dysfunction in mice homozygous for the shaker 2 and shaker 2(J) alleles. The shaker 2 allele is a previously described missense mutation of a highly conserved residue in the motor domain of myosin XV. The shaker 2(J) lesion, in contrast, is a 14.7 kb deletion that removes the last six exons from the 3"-terminus of the Myo15 transcript. These exons encode a FERM (F, ezrin, radixin and moesin) domain that may interact with integral membrane proteins. Despite the deletion of six exons, Myo15 mRNA transcripts and protein are present in the post-natal day 1 shaker 2(J) inner ear, which suggests that the FERM domain is critical for the development of normal hearing and balance. Myo15 transcripts are first detectable at embryonic day 13.5 in wild-type mice. Myo15 transcripts in the mouse inner ear are restricted to the sensory epithelium of the developing cristae ampularis, macula utriculi and macula sacculi of the vestibular system as well as to the developing organ of Corti. Both the shaker 2 and shaker 2(J) alleles result in abnormally short hair cell stereocilia in the cochlear and vestibular systems. This suggests that Myo15 may be important for both the structure and function of these sensory epithelia.

Alleles↗
PubMed2000

The otoconia of the guinea pig utricle: internal structure, surface exposure, and interactions with the filament matrix.

A unique feature of the vertebrate gravity receptor organs, the saccule and utricle, is the mass of biomineral structures, the otoconia, overlying a gelatinous matrix also called "otoconial membrane" on the surface of the sensory epithelium. In mammals, otoconia are deposits of calcium carbonate in the form of composite calcite crystals. We used quick-freezing, deep etching to examine the otoconial mass of the guinea pig utricle. The deep-etching step exposed large expanses of intact and fractured otoconia, showing the fine structure and relationship between their internal crystal structure, their surface components, and the filament matrix in which they are embedded. Each otoconium has a compact central core meshwork of filaments and a composite outer shell of ordered crystallites and macromolecular aggregates. A distinct network of 20-nm beaded filaments covers the surface of the otoconia. The otoconia are interconnected and secured to the gelatinous matrix by surface adhesion and by confinement within a loose interotoconial filament matrix. The gelatinous matrix is a dense network made of yet another type of filament, 22 nm in diameter, which are cross-linked by shorter filaments, characteristically 11 nm in diameter. Our freeze-etching data provide a structural framework for considering the molecular nature of the components of the otoconial complex, their mechanical properties, and the degree of biological versus chemical control of otoconia biosynthesis.

Animals↗
PubMed2000

Modifier genes of hereditary hearing loss.

Phenotypic variation between individuals with the same disease alleles may be attributable to the genotype at another locus, which is referred to as a modifier gene. Recent functional studies of modifier genes of hearing-loss loci have begun to refine our understanding of hearing processes and will guide the rational design of medical therapies for hearing loss.

Alleles↗
PubMed1999

CNS myelin and sertoli cell tight junction strands are absent in Osp/claudin-11 null mice.

Oligodendrocyte-specific protein (OSP)/claudin-11 is a recently identified transmembrane protein found in CNS myelin and testis with unknown function. Herein we demonstrate that Osp null mice exhibit both neurological and reproductive deficits: CNS nerve conduction is slowed, hindlimb weakness is conspicuous, and males are sterile. Freeze fracture reveals that tight junction intramembranous strands are absent in CNS myelin and between Sertoli cells of mutant mice. Our results demonstrate that OSP is the mediator of parallel-array tight junction strands and distinguishes this protein from other intrinsic membrane proteins in tight junctions. These novel results provide direct evidence of the pivotal role of the claudin family in generating the paracellular physical barrier of tight junctions necessary for spermatogenesis and normal CNS function.

Animals↗
PubMed1999

Characterization of the human and mouse unconventional myosin XV genes responsible for hereditary deafness DFNB3 and shaker 2.

Mutations in myosin XV are responsible for congenital profound deafness DFNB3 in humans and deafness and vestibular defects in shaker 2 mice. By combining direct cDNA analyses with a comparison of 95.2 kb of genomic DNA sequence from human chromosome 17p11.2 and 88.4 kb from the homologous region on mouse chromosome 11, we have determined the genomic and mRNA structures of the human (MYO15) and mouse (Myo15) myosin XV genes. Our results indicate that full-length myosin XV transcripts contain 66 exons, are >12 kb in length, and encode 365-kDa proteins that are unique among myosins in possessing very long approximately 1200-aa N-terminal extensions preceding their conserved motor domains. The tail regions of the myosin XV proteins contain two MyTH4 domains, two regions with similarity to the membrane attachment FERM domain, and a putative SH3 domain. Northern and dot blot analyses revealed that myosin XV is expressed in the pituitary gland in both humans and mice. Myosin XV transcripts were also observed by in situ hybridization within areas corresponding to the sensory epithelia of the cochlea and vestibular systems in the developing mouse inner ear. Immunostaining of adult mouse organ of Corti revealed that myosin XV protein is concentrated within the cuticular plate and stereocilia of cochlear sensory hair cells. These results indicate a likely role for myosin XV in the formation or maintenance of the unique actin-rich structures of inner ear sensory hair cells.

Alternative Splicing↗
PubMed1999

ATP-Induced Ca(2+) release in cochlear outer hair cells: localization of an inositol triphosphate-gated Ca(2+) store to the base of the sensory hair bundle.

We used a high-performance fluorescence imaging system to visualize rapid changes in intracellular free Ca(2+) concentration ([Ca(2+)](i)) evoked by focal applications of extracellular ATP to the hair bundle of outer hair cells (OHCs): the sensory-motor receptors of the cochlea. Simultaneous recordings of the whole-cell current and Calcium Green-1 fluorescence showed a two-component increase in [Ca(2+)](i). After an initial entry of Ca(2+) through the apical membrane, a second and larger, inositol triphosphate (InsP(3))-gated, [Ca(2+)](i) surge occurred at the base of the hair bundle. Electron microscopy of this intracellular Ca(2+) release site showed that it coincides with the localization of a unique system of endoplasmic reticulum (ER) membranes and mitochondria known as Hensen's body. Using confocal immunofluorescence microscopy, we showed that InsP(3) receptors share this location. Consistent with a Ca(2+)-mobilizing second messenger system linked to ATP-P2 receptors, we also determined that an isoform of G-proteins is present in the stereocilia. Voltage-driven cell shape changes and nonlinear capacitance were monitored before and after ATP application, showing that the ATP-evoked [Ca(2+)](i) rise did not interfere with the OHC electromotility mechanism. This second messenger signaling mechanism bypasses the Ca(2+)-clearance power of the stereocilia and transiently elevates [Ca(2+)](i) at the base of the hair bundle, where it can potentially modulate the action of unconventional myosin isozymes involved in maintaining the hair bundle integrity and potentially influence mechanotransduction.

Adenosine Triphosphate↗
PubMed1999

Redistribution and phosphorylation of occludin during opening and resealing of tight junctions in cultured epithelial cells.

We studied the expression, distribution, and phosphorylation of the tight junction (TJ) protein occludin in confluent MDCK cell monolayers following three procedures for opening and resealing of TJs. When Ca(2+) is transiently removed from the culture medium, the TJs open and the cells separate from each other, but the occludin band around each cell is retained. When Ca(2+) is reintroduced, the TJs reseal. When the monolayers are exposed to prolonged Ca(2+) starvation the cells maintain contact, but occludin disappears from the cell borders and can be detected only in a cytoplasmic compartment. When Ca(2+) is reintroduced, new TJs are assembled and the transepithelial electrical resistance (TER) is reestablished in about 20 hr. Monolayers treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) show a different pattern of TJ opening: the cell-cell contact is maintained but the TJ strand network, as seen in freeze-fracture replicas, becomes discontinuous. Occludin is still localized at the cell periphery, but in a pattern of distribution that matches the discontinuous TJ. These TJs do not reseal even 24 hr after removal of the TPA. Western blot analysis showed that the 62-65 kD double band of occludin did not change with these treatments. However, in vivo phosphorylation analysis showed that the TPA treatment reduced the phosphorylation levels of occludin, while the prolonged Ca(2+) starvation completely dephosphorylated the two occludin bands. In addition, a highly phosphorylated 71 kD band that immunoprecipitates with occludin is not present when TJ is opened by the Ca(2+) removal. Phosphoaminoacid analysis showed that the 62-65 kD occludin bands are phosphorylated on serine and threonine, while the 71 kD band was phosphorylated exclusively on serine. Our results provide further evidence that phosphorylation of occludin is an important step in regulating TJ formation and permeability.

Animals↗
PubMed1999

Small synthetic peptides homologous to segments of the first external loop of occludin impair tight junction resealing.

This study shows that resealing of opened tight junctions (TJs) is impaired by interaction with oligopeptides homologous to the external domain of chick occludin. The experiments were carried out with confluent A6 cell monolayers grown on collagen supports under stable transepithelial electrical resistance (TER). The monolayers were bathed on the apical side with a 75 mm KCl solution and on the basolateral side by NaCl-Ringer's solution. TJ opening was induced by basolateral Ca2+ removal and was characterized by a marked drop of TER. The reintroduction of Ca2+ triggered junction resealing as indicated by an elevation of TER to control values. Custom-made peptides SNYYGSGLSY (corresponding to the residues 100 to 109) and SNYYGSGLS (residues 100 to 108), homologous to segments of the first external loop of chick occludin molecule, impaired junction resealing when the peptides were included in the apical bathing fluid (concentrations in the range of 0.5 to 1.5 mg/ml). Peptide removal from the apical solution usually triggered a slow recovery of TER, indicating a slow recovery of the TJ seal. Changes in localization of ZO-1, a cytoplasmic protein that underlies the membrane at the TJs, were evaluated immunocytochemically following Ca2+ removal and reintroduction. The presence or absence of the oligopeptides showed no influence on the pattern of change of ZO-1 localization. These observations support the hypothesis that the TJ seal results from the interaction of specific homologous segments of occludin on the surface of adjacent cells. Additionally, our results show that small peptides homologous to segments of the occludin first external loop can be used as specific reagents to manipulate the permeability of tight junctions.

Animals↗
PubMed1999

Three-dimensional analysis of the 16 nm urothelial plaque particle: luminal surface exposure, preferential head-to-head interaction, and hinge formation.

The luminal surface of mouse urothelium in contact with the urine is almost entirely covered with plaques consisting of uroplakin-containing particles that form p6 hexagonal crystals with a center-to-center distance of 16 nm. A combination of quick-freeze/deep-etch images and our previous negative staining data indicate that the head domain of the uroplakin particle, which is exposed without an extensive glycocalyx shield, interacts closely with the head domains of the neighboring particles, while the membrane-embedded tail domains are farther apart; and that urothelial particles and plaques are not rigid structures as they can change their configuration in response to mechanical perturbations. Based on these data, we have constructed three-dimensional models depicting the structural organization of urothelial particles and plaques. Our models suggest that the head-to-head interaction may play a key role in determining the shape and size of the urothelial plaques. These models can explain many properties of urothelial plaques including their unique shape, detergent-insolubility, and morphological changes during vesicle maturation.

Animals↗
PubMed1999

Establishment and characterization of conditionally immortalized organ of corti cell lines.

A culture of cells was isolated from the organ of Corti of 2-week-old H-2Kb-tsA58 (Immortomouse) transgenic mice. All cells of these mice harbor a mutant of the simian virus 40 A-gene, encoding a thermolabile large T-antigen (Tag) protein. At 33 degrees C the Tag protein is functional and induces cell proliferation, but at 39 degrees C it is rapidly denatured and inactivated. Isolated organ of Corti cells growing at 33 degrees C were predominantly small, rounded or fusiform and proliferated rapidly. When moved to 39 degrees C, the cells reduced their rate of proliferation and differentiated into specific morphological phenotypes. Four cell lines were cloned by limiting dilution and characterized by immunofluorescence microscopy and Western blot. The cell lines, named OC-k1, OC-k2, OC-k3 and OC-k4, have been passaged at least 50 times with retention of a stable phenotype. These cell lines were all positive for the neuroepithelial precursor cell marker nestin and for the inner ear cell marker OCP2. In addition, the cells showed reactivity to epithelial and neuronal cell markers, but with a pattern of protein expression different for each clone and different between cells of the same clone growing at 33 degrees C or 39 degrees C. Some of the clones exhibited asymmetric cell division which is a characteristic commonly ascribed to stem cells. These cell lines can be used advantageously to study mechanisms and signals involved in the control of cell differentiation and morphogenesis of the mammalian inner ear and to isolate inner ear specific proteins.

Animals↗
PubMed1999

Atomic force microscopy study of tooth surfaces.

Atomic force microscopy (AFM) was used to study tooth surfaces in order to compare the pattern of particle distribution in the outermost layer of the tooth surfaces. Human teeth and teeth from a rodent (Golden hamster), from a fish (piranha), and from a grazing mollusk (chiton) with distinct feeding habits were analyzed in terms of particle arrangement, packing, and size distribution. Scanning electron microscopy and transmission electron microscopy were used for comparison. It was found that AFM gives high-contrast, high-resolution images and is an important tool as a source of complementary and/or new structural information. All teeth were cleaned and some were etched with acidic solutions before analysis. It was observed that human enamel (permanent teeth) presents particles tightly packed in the outer surface, whereas enamel from the hamster (continuously growing teeth) shows particles of less dense packing. The piranha teeth have a thin cuticle covering the long apatite crystals of the underlying enameloid. This cuticle has a rough surface of particles that have a globular appearance after the brief acidic treatment. The similar appearance of the in vivo naturally etched tooth surface suggests that the pattern of globule distribution may be due to the presence of an organic material. Elemental analysis of this cuticle indicated that calcium, phosphorus, and iron are the main components of the structure while electron microdiffraction of pulverized cuticle particles showed a pattern consistent with hydroxyapatite. The chiton mineralized tooth cusp had a smooth surface in an unabraded region and a very rough structure with the magnetite crystals (already known to make part of the structure) protruding from the surface. It was concluded that the structures analyzed are optimized for efficiency in feeding mechanism and life span of the teeth.

Acids↗
PubMed1999

Structural basis for mechanical transduction in the frog vestibular sensory apparatus: III. The organization of the otoconial mass.

The saccule and the utricle of the vestibular system detect linear acceleration and gravity. Sensory transduction in these organs depends on myriads of calcium carbonate crystals of high specific gravity, called otoconia, embedded in a filament matrix that overlies the sensory epithelium. The coexistence of hard crystals and slender filaments in this complex extracellular matrix makes it difficult to analyze by conventional electron microscopy. We have now examined this structure in the bullfrog saccule using the quick-freeze, deep-etch replica technique. The otoconia in their typical aragonite polymorph shape exhibit smooth surfaces and are embedded in a loose matrix made of two types of filaments. The regular surface of the otoconia forms a natural smooth background against which we could observe with unprecedented detail the network organization and substructure of the filaments. One type of filament is 8 nm in diameter, while the other, which has a characteristic beaded appearance, is 15 nm in diameter. Both types of filaments either make lateral connections with or end directly on the surface of the otoconia. A consistent observation was the presence of short filaments that directly cross-link adjacent otoconia. Very few otoconia were fractured in an orientation that would allow the study of their internal architecture. These otoconia presented a typical conchoidal cleavage of aragonite. Although crystallites were not clearly apparent, thin lamellar microstructures appeared oriented both perpendicularly and longitudinally to the major otoconial axis. This structural study establishes a framework for the identification of the molecular components present in this unique extracellular matrix and may also help elucidate their role in mechanical transduction.

Animals↗
PubMed1999

Urothelial hinge as a highly specialized membrane: detergent-insolubility, urohingin association, and in vitro formation.

Urothelial surface is covered by numerous plaques (consisting of asymmetric unit membranes or AUM) that are interconnected by ordinary looking hinge membranes. We describe an improved method for purifying bovine urothelial plaques using 2% sarkosyl and 25 mM NaOH to remove contaminating membrane and peripheral proteins selectively. Highly purified plaques interconnected by intact hinge areas were obtained, indicating that the hinges are as detergent-insoluble as the plaques. These plaque/hinge preparations contained uroplakins, an as yet uncharacterized 18-kDa plaque-associated protein, plus an 85-kDa glycoprotein that is known to be hinge-associated in situ. Examination of the isolated, in vitro-resealed bovine AUM vesicles by quick-freeze deep-etch showed that each AUM particle consists of a 16-nm, luminally exposed "head" anchored to the lipid bilayer via a 9-mm transmembranous "tail", and that an AUM plaque can break forming several smaller plaques separated by newly formed particle-free, hinge-like areas. These data lend support to our recently proposed three-dimensional model of mouse urothelial plaques. In addition, our findings suggest that urothelial plaques are dynamic structures that can rearrange giving rise to new plaques with intervening hinges; that the entire urothelial apical surface (both plaque and hinge areas) is highly specialized; and that these two membrane domains may be equally important in fulfilling some of the urothelial functions.

Animals↗
PubMed1999

Structural elements common to mitosis and apoptosis.

Both mitotic and apoptotic cells display hypercondensation of the chromatin and loss of the nuclear envelope (Lazebnik et al., 1993). Herein, we describe a third similarity between the two processes. We have observed, initially in apoptotic cells of the PC-12 lineage clusters of 40-60 (approximately 50) nm vesicles adjoined by a minor contingent of tubule vesicular elements of 100-200 nm which are indistinguishable from their vesicular counterparts in mitotic PC-12 cells. The clusters of approximately 50 nm vesicles were subsequently observed in all studied rat tissue cells in apoptosis (plasma cells and macrophages, secretory epithelial cells from pancreatic acini, ventral lobe of prostate and mammary gland). Clusters of approximately 50 nm vesicles comparable to those of the PC-12 cells were found in HeLa cells treated with human alfa TNF, in WEHI-3 cells exposed to VM 26 (a teneposide) (Sesso et al., 1997) and in HL-60 cells treated with thapsigargin. PC-12 and HeLa cells affixed to coverslips were double labelled and examined with the fluorescence microscope to reveal simultaneously the disposition of the chromatin with Hoechst stain and the distribution of the fluorescence of Golgi or of Golgi-associated proteins. A common pattern of fluorescence was observed in a minor proportion of apoptotic cells using three different antibodies used. The label frequently appeared as finely dispersed granules in the cytoplasm. In some apoptotic cells, relatively coarse granules were observed. This pattern of label distribution is compatible with the disposition of vesicular clusters we have encountered in apoptotic PC-12 cells sectioned serially or semi serially. In such sections of both mitotic and apoptotic PC-12 cells, we noticed that the conglomerates of 50 nm vesicles were frequently associated with cisternae of the rough ER. Vesicles of similar size were also noted pinching off from the extremities of Golgi cisternae reduced in size. These cisternae diminish in length and width when they are in the process of disassembling at the very beginning of mitosis and in apoptosis.

Animals↗
PubMed1998

Localization of myosin-V in the centrosome.

The perinuclear localization of myosin-V was investigated in a variety of cultured mammalian cells and in primary cultures of rat hippocampus. In all cells investigated, myosin-V immunoreactivity was associated with the centrosome. In interphase cells, myosin-V was found in pericentriolar material, and in both mother and daughter centrioles. These results were obtained by using two different fixation protocols with three different affinity-purified antibodies that recognized a single band in Western blots. During cell division, myosin-V staining was intense throughout the cytoplasm and was concentrated in a trail between migrating centrioles and in the mitotic spindle poles and spindle fibers. The centrosome targeting site was determined to reside within the globular tail domain, because centrosome association also was observed in living cells transfected with DNA encoding the tail domain fused with a green fluorescent protein tag, but not in cells transfected with the vector encoding green fluorescent protein by itself.

Animals↗
PubMed1998

Presynaptic localization of G protein isoforms in the efferent nerve terminals of the mammalian cochlea.

Heterotrimeric guanine nucleotide binding proteins (G proteins) are known to be involved in receptor-mediated synaptic activity. In order to determine which G protein isoforms, if any, are involved in synaptic regulation in the organ of Corti, we performed an extensive immunocytochemical screening. We localized a Galpha(q/11) isoform to the efferent nerve terminals using antibodies specific against the alpha subunit of these proteins. The label was observed in the efferent boutons contacting either the outer hair cells or the afferent fibers at the inner spiral bundle. We compared the localization of this isoform to that of the presynaptic protein SNAP-25 in double labeling experiments. Galpha(q/11) immunoreactivity was present predominantly in the cytoplasm of the presynaptic boutons in a region of high density of synaptic vesicles, while SNAP-25 was localized predominantly in the plasma membrane of the boutons. No label for these proteins was found at the afferent synapses, including the presynaptic terminals on hair cells. These results suggest that an isoform of the Gq subfamily of the G proteins might be involved in presynaptic modulation of neurotransmitter release at the cochlear efferents.

Animals↗