Ultrastructural localisation of spectrin in sensory and supporting cells of guinea-pig organ of Corti

Spectrin is a cytoskeletal protein found in the cortex of many cell types. It is known to occur in cochlear outer hair cells (OHCs) with previous immunoelectron microscopical studies showing that it is located in the cuticular plate and the cortical lattice. The latter is a network of filaments associated with the lateral plasma membrane that is thought to play a role in OHC motility. Spectrin has also been found in inner hair cells (IHCs) and supporting cells using immunofluorescent techniques, but its ultrastructural distribution in these cells has not yet been described. This has, therefore, been investigated using a monoclonal antibody to alpha-spectrin in conjunction with pre- and post-embedding immunogold labelling for transmission electron microscopy. Labelling was found in a meshwork of filaments beneath the plasma membranes of both IHCs and supporting cells and, in pillar cells, close to microtubule/microfilament arrays. It was also found in association with the stereocilia of OHCs and IHCs and, as expected, in the cortical lattice and cuticular plate of OHCs. Thus, spectrin is a general component of cytoskeletal structures involved in maintaining the specialised cell shapes in the organ of Corti and may contribute to the mechanical properties of all the cell types examined.     

Analysis of the micropipet experiment with the anisotropic outer hair cell wall

The outer hair cell makes both passive and active contributions to basilar membrane mechanics. The outer hair cell mechanics is strongly coupled to the elastic properties of the cell lateral wall. The lateral wall experiences both in-plane deformations and bending under physiological and experimental conditions. To characterize the outer hair cell wall, the model of an orthotropic cylindrical shell is used. The elastic constants of the wall are estimated by solving a set of three equations based on the analyses of three independent experiments. The first equation is derived from a new interpretation of the micropipet experiment; the other two are obtained from the axial loading and the osmotic challenge experiments. The two Young's moduli corresponding to the longitudinal and circumferential directions and two Poisson's ratios are estimated. The longitudinal, circumferential, and mixed modes of the bending stiffness are also estimated. The sensitivity of the derived constants to the variation of the cell axial stiffness, which has been measured by several independent groups, is examined. The new estimates are also compared with results obtained by using the assumption of the wall isotropy.     

A dynamic model of outer hair cell motility including intracellular and extracellular fluid viscosity

The deformation response of a guinea pig outer hair cell is modeled for mechanical and electrical stimulation up to 25 kHz. The analysis uses a Fourier series technique for a finite length cell surrounded internally and externally by a much larger continuum of viscous fluid. The analytical solution predicts that outer hair cell length changes occur due to applied mechanical or electrical stimulation without significant resonance, characteristic of a highly damped system. The deformation is found to have little attenuation up to a corner frequency of about 2 kHz for long cells and 10 kHz for short cells, in agreement with published experimental results. For electrical loading of 1 mV across the lateral cell wall, deformation for short cells is calculated to be greater than 1 nm for frequencies up to 20 kHz. These results support the proposition that in vivo the outer hair cell modifies the character of basilar membrane deformation on a cycle-by-cycle basis. An estimate of the capability of the cell to supply energy to the basilar membrane is given based on published values of outer hair cell material properties.     

Mapping the distribution of outer hair cell voltage-dependent conductances by electrical amputation

The mammalian outer hair cell (OHC) functions not only as sensory receptor, but also as mechanical effector; this unique union is believed to enhance our ability to discriminate among acoustic frequencies, especially in the kilohertz range. An electrical technique designed to isolate restricted portions of the plasma membrane was used to map the distribution of voltage-dependent conductances along the cylindrical extent of the cell. We show that three voltage-dependent currents, outward K, I(K,n), and I(Ca) are localized to the basal, synaptic pole of the OHC. Previously we showed that the lateral membrane of the OHC harbors a dense population of voltage sensor-motor elements responsible for OHC motility. This segregation of membrane molecules may have important implications for auditory function. The distribution of OHC conductances will influence the cable properties of the cell, thereby potentially controlling the voltage magnitudes experienced by the motility voltage sensors in the lateral membrane, and thus the output of the "cochlear amplifier."     

A kinase anchor protein 75 targets regulatory (RII) subunits of cAMP-dependent protein kinase II to the cortical actin cytoskeleton in non-neuronal cells

Neuronal A kinase anchor protein (AKAP) homologs, such as AKAPs 75 and 150, tether cAMP-dependent protein kinase II (PKAII) isoforms to the postsynaptic cytoskeleton, thereby creating target sites for cAMP action. These AKAPs, which bind regulatory subunits (RIIs) of PKAII, are also expressed in certain non-neuronal cells. Non-neuronal cell lines that stably express wild type and mutant AKAP75 transgenes were generated to investigate the extraneuronal function of AKAPs. In non-neuronal cells, AKAP75 accumulates selectively in the actin-rich, cortical cytoskeleton in close proximity with the plasma membrane. AKAP75 efficiently sequesters cytoplasmic RIIalpha and RIIbeta (PKAII isoforms) and translocates these polypeptides to the cell cortex. Two structural modules in AKAP75, T1 (residues 27-48), and T2 (residues 77-100), are essential for targeting AKAP75.RII complexes to the cortical cytoskeleton. Deletions or amino acid substitutions in T1 and/or T2 result in the dispersion of both AKAP75 and RII subunits throughout the cytoplasm. AKAP75 is co-localized with F-actin and fodrin in the cortical cytoskeleton. Incubation of cells with 5 microM cytochalasin D disrupts actin filaments and dissociates actin from the cell cortex. In contrast, the bulk of AKAP75 and fodrin remain associated with the cortical region of cytochalasin D-treated cells. Thus, targeting of AKAP75 does not depend upon direct binding with F-actin. Rather, AKAP75 (like fodrin) may be associated with a multiprotein complex that interacts with integral plasma membrane proteins.     

Correspondence between middle frequency auditory loss in vivo and outer hair cell shortening in vitro

The aromatic hydrocarbon, toluene, has been reported to disrupt auditory system function both in occupational epidemiological and in laboratory animal investigations. This agent, along with several other organic solvents, impairs hearing preferentially at middle frequencies - a finding that distinguishes these agents from the traditional high frequency impairment observed with ototoxic drugs such as aminoglycoside antibiotics and cisplatin. Prior investigations performed in vivo have identified the outer hair cell as a probable target for toluene exposure. The purpose of this investigation was to determine directly whether outer hair cells isolated from the guinea pig cochlea show morphological alterations consistent with the toxic response seen in physiological studies with toluene exposure. The effect of toluene superfusion on outer hair cell shortening was assessed for cells harvested from different locations within the cochlea. Control studies included assessment of cell shortening among outer hair cells exposed to trimethyltin and cells exposed to benzene. Trimethyltin disrupts high frequency hearing preferentially and benzene does not produce hearing loss in vivo. Toluene at a concentration of 100 microM produced a marked shortening of outer hair cells although the effect was significantly greater among cells isolated from the apical half of the cochlea than from the basal half of the cochlea. By contrast, trimethyltin at the same concentration produced a preferential shortening among outer hair cells from the base of the cochlea. Benzene (100 microM) did not disrupt outer hair cell length of cells harvested from the apex. The results indicate that intrinsic features of outer hair cells contribute significantly to the site of ototoxic impairment observed in vivo for toluene.     

Current noise spectrum and capacitance due to the membrane motor of the outer hair cell: theory

The voltage-dependent motility of the outer hair cell is based on a membrane motor densely distributed in the lateral membrane. The gating charge of the membrane motor is manifested as a bell-shaped membrane potential dependence of the membrane capacitance. In this paper it is shown that movements of the gating charge should produce a high-pass current noise described by an inverse Lorentzian similar to the one shown by Kolb and L?uger for ion carriers. The frequency dependence of the voltage-dependent capacitance is also derived. These derivations are based on membrane motor models with two or three states. These two models lead to similar predictions on the capacitance and current noise. It is expected that the examination of the spectral properties of these quantities would be a useful means of determining the relaxation time for conformational transitions of the membrane motor.     

The localization of synaptophysin in the organ of Corti of the human as shown by immunoelectron microscopy

Synaptophysin, or p38, a polypeptide of molecular weight 38 kD, is a calcium-binding membrane protein found in synaptic vesicles of neurons and smooth surfaced vesicles of neuroendocrine cells. Six human neonatal and infant temporal bones were fixed in paraformaldehyde and glutaraldehyde, decalcified in EDTA and were than immunoreacted for synaptophysin (ICN Biomedicals) using the avidin-biotin reaction (ABC kit, Vector Labs). The tissue was then prepared for light microscopic surface preparation, radial sections of 5 microns, and serial section electron microscopy. At a light microscopic level, the inner spiral bundle, tunnel spiral bundle, upper tunnel crossing fibers and the base of outer hair cells were stained. At the base of outer hair cells, the immunoreactivity was seen to decrease from the base to the apex and from the first to third outer hair cells. At an electron microscopic level, immunoreactivity at the base of outer hair cells was limited to vesiculated efferent fibers. The degree of immunoreactivity between adjacent efferent fibers varied significantly. Immunoreactive vesiculated endings were also found in the supranuclear region of outer hair cells.     

Role of calpain on hypoxic myocyte injury

The calcium-activated neutral protease, calpain, was tested for its proteolytic effects to degrade the cell membrane spectrin-like protein, fodrin, during hypoxia. Cardiac myocytes, isolated from neonatal rat hearts, were incubated under hypoxic conditions for 6 hours. The cell death during hypoxia rose to 80% after 6 hours. Extracellular protease activity was much more elevated during hypoxia, than in aerobic states at 6 hours. Intracellular protease activity was also elevated in hypoxia. These protease activities were markedly inhibited by the cysteine protease inhibitor E-64 and the calpain specific inhibitor, calpastatin. Hypoxic cell death was also suppressed by E-64. Cell membrane proteins prepared from hypoxic myocytes were examined with electroblots stained for fodrin by the peroxidase method. A 125 kilodalton immunoreactive degradation product of fodrin was found under hypoxic conditions. Treatment with E-64 inhibited both the appearance of this degradation band and the decrease in the content of fodrin. These observations indicate that calpain is activated during hypoxia and that it is related to cell membrane protein degradation, especially in fodrin. The data also suggest that protease inhibitor E-64 treatment may be beneficial in protection against hypoxic myocyte injury.     

Can the neurotrophic hypothesis explain degeneration and loss of plasticity in mature and ageing autonomic nerves?

The development of Ca-ATPase immunoreactivity in gerbil outer hair cells (OHCs), assayed by immunofluorescence and postembedding immunocytochemistry, is reported here. In the adult, a linear array of label is seen inside the lateral plasma membrane. The ultrastructural distribution of Ca-ATPase near the OHC lateral plasma membrane was examined using immunogold cytochemistry and showed this calcium pumping enzyme to be present throughout the subsurface cisternal complex (SSC), especially near the innermost layers. During development, Ca-ATPase immunoreactivity appeared in patches near the lateral plasma membrane of some OHCs of the third row by 12 days after birth (DAB). By 15-16 DAB, punctate immunoreactivity was detected in the second and first rows. At 20 DAB, immunostaining near OHC lateral plasma membrane was increased, but was less continuous than OHC staining in the adult cochlea. The appearance of Ca-ATPase in OHCs coincides with the onset of auditory function and isolated OHC motility in the gerbil. The ultrastructural demonstration of abundant sites of calcium pumps in the SSC supports a role for this structure in the intracellular storage of calcium. These findings suggest a possible role of Ca-ATPase and the SSC in the regulation of slow motility of OHCs which has been reported to depend on intracellular calcium concentration.     

Compartmentalized vesicular traffic around the hair cell cuticular plate

Through thin-section and freeze-fracture electron microscopy, we identify structural correlates of an intense vesicular traffic in a narrow band of cytoplasm around the cuticular plate of the bullfrog vestibular hair cells. Myriads of coated and uncoated vesicles associated with longitudinally oriented microtubules populate the narrow cytoplasmic region between the cuticular plate and the actin network of the apical junctional belt. If microtubules in the sensory hair cells, like those in axons, are pathways for organelle transport, then the characteristic distribution of microtubules around the cuticular plate represents transport pathways across the apical region of the hair cells. This compartmentalized membrane traffic system appears to support an intense vesicular release and uptake along a band of apical plasma membrane near the cell border. Functions of this transport system may include membrane recycling as well as exocytotic and endocytotic exchange between the hair cell cytoplasm and the endolymphatic compartment.     

Tunnel crossing fibers and their synaptic connections within the inner hair cell region in the organ of corti in the maturing mouse

Combined ultrastructural and immunocytochemical studies reveal that in the adolescent 12- to 17-day-old mouse the afferent tunnel crossing fibers that innervate outer hair cells receive synaptic contacts from three distinct sources: the GABAergic fibers (GABA = gamma-aminobutyric acid) of the lateral olivocochlear bundle, the non-GABAergic efferent tunnel crossing fibers, and the inner hair cells themselves. The GABAergic fibers give off collaterals that synapse with the afferent tunnel fibers as they cross the inner hair cell region. These collaterals also form synapses with afferent radial dendrites that are synaptically engaged with the inner hair cells. Vesiculated varicosities of non-GABAergic efferent tunnel fibers also synapse upon the outer spiral afferents. Most of this synaptic activity occurs within the inner pillar bundle. Distinctive for this region are synaptic aggregations in which several neuronal elements and inner hair cells are sequentially interconnected. Finally, most unexpected were the afferent ribbon synapses that inner hair cells-formed en passant on the shafts of the apparent afferent tunnel fibers. The findings indicate that: (1) the afferent tunnel (i.e., outer spiral) fibers may be postsynaptic to both the inner and the outer hair cells; (2) the non-GABAergic efferent and the afferent tunnel fibers form extensive synaptic connections before exiting the inner pillar bundle; (3) the GABAergic component of the lateral olivocochlear system modulates synaptically both radial and outer spiral afferents.     

Pathologic mechanoelectric transduction of outer hair cells as the cause of recruitment

It is believed that the sound-induced travelling wave in the mammalian cochlea is enhanced and sharpened by a positive feedback mechanism. This causes the passive linear basilar membrane growth function to become non-linear. The present paper shows that nonlinear basilar membrane vibration is due to the nonlinear growth function of the receptor potential of outer hair cells, which can be described by a 2nd-order Boltzmann function. Since intensity coding in the inner ear depends on an interaction of nonlinear basilar membrane motion and nerve fibers with three different types of synaptic threshold and growth function, the process is directly dependent on an intact mechanoelectrical transduction of outer hair cells. According to the proposed model, a loss in efficiency of outer hair cell mechanoelectrical transduction must lead to both a reduction in gain (i.e., hearing loss) and a linearizing of the response. As a result, once above threshold, the changes of stereociliary displacement, basilar membrane displacement and neural firing rate per unit change of sound intensity must be larger than for the healthy cochlea with its compressive nonlinearity.     

Evidence for differential regulation of calcium by outer versus inner hair cells: plasma membrane Ca-ATPase gene expression

The expression of mRNA encoding plasma membrane calcium ATPase (PMCA) subunit isoforms (1-4) and splice variants was examined in the adult and developing rat cochlea by PCR and in situ hybridization. High levels of PMCA mRNA expression were observed in the neurons of the spiral ganglion, and in hair cells. Spiral ganglion neurons expressed PMCA 1-3 beginning in embryonic development, reaching high levels shortly after birth, and continuing into adulthood. Inner hair cells expressed PMCA 1 at moderate levels from birth to the time of onset of cochlear function on postnatal day 12, and strongly from then until adulthood. Outer hair cells expressed PMCA 2 at high levels from shortly after birth through adulthood. The data suggest that the calcium clearance requirements of inner and outer hair cells are distinct. PMCA 2 is the isoform with the highest affinity for calmodulin, and has also been associated with high levels of inositol triphosphate. Its presence in outer hair cells suggests that regulation of the enzyme by calmodulin may be particularly important for this hair cell type. It further suggests that inositol phosphate may play a unique role in the outer hair cell.     

Cochlear outer hair cell bending in an external electric field

We have used a high-resolution motion analysis system to reinvestigate shape changes in isolated guinea pig cochlear outer hair cells (OHCs) evoked by low-frequency (2-3 Hz) external electric stimulation. This phenomenon of electromotility is presumed to result from voltage-dependent structural changes in the lateral plasma membrane of the OHC. In addition to well-known longitudinal movements, OHCs were found to display bending movements when the alternating external electric field gradients were oriented perpendicular to the cylindrical cell body. The peak-to-peak amplitude of the bending movement was found to be as large as 0.7 microm. The specific sulfhydryl reagents, p-chloromercuriphenylsulfonic acid and p-hydroxymercuriphenylsulfonic acid, that suppress electrically evoked longitudinal OHCs movements, also inhibit the bending movements, indicating that these two movements share the same underlying mechanism. The OHC bending is likely to result from an electrical charge separation that produces depolarization of the lateral plasma membrane on one side of the cell and hyperpolarization on the other side. In the cochlea, OHC bending could produce radial distortions in the sensory epithelium and influence the micromechanics of the organ of Corti.     

Does the organ of Corti attempt to differentiate new hair cells after antibiotic intoxication in rat pups?

In the adult mammalian cochlea, post-injury hair cell losses are considered to be irreversible. Recent studies in cochlear explants of embryonic rodents show that the organ of Corti can replace lost hair cells after injury. We have investigated this topic in vivo during the period of cochlear development. Rat pups were treated with a daily subcutaneous injection of 500 mg/kg amikacin for eight consecutive days between postnatal day 9 (PND 9) and PND 16. During this period the organ of Corti is not fully mature, but hair cells are hyper-sensitive to aminoglycoside antibiotics. Scanning and transmission electron microscopy was used to evaluate morphological changes in the organs of Corti during the treatment and at different post-treatment periods, up until PND 90. A massive loss in outer and inner hair cells was observed at least as early as PND 14. A prominent feature in the apical part of cochleas at PND 21 and 35 was the transient presence of small atypical cells in the region of pre-existing outer hair cells. These atypical cells had tufts of microvilli reminiscent of nascent stereociliary bundles. A second striking observation was the replacement of degenerating inner hair cells by pear-shaped supporting cells throughout the cochlea. These cells were covered with long microvilli, and their basal pole was contacted by both afferent and efferent fibers, as in the early stages of inner hair cell maturation. At PND 55 and 90, these features were not clearly observed due to further cytological changes in the organ of Corti. It is possible that an attempt at hair cell neodifferentiation could occur in vivo after an amikacin treatment in the rat during the period of cochlear hyper-sensitivity to antibiotic.     

Programmed cell death in the root cortex of soybean root necrosis mutants

The soybean root necrosis (rn) mutation causes a progressive browning of the root soon after germination that is associated with accumulation of phytoalexins and pathogenesis-related proteins and an increased tolerance to root-borne infection by the fungal pathogen, Phytophthora sojae. Grafting and decapitation experiments indicate that the rn phenotype is root-autonomous at the macroscopic level. However, the onset and severity of browning was modulated in intact plants by exposure to light, as was the extent of lateral root formation, suggesting that both lateral roots and the rn phenotype could be directly or indirectly controlled by similar shoot-derived factors. Browning first occurs in differentiated inner cortical cells adjacent to the stele and is preceded by a wave of autofluorescence that emanates from cortical cells opposite the xylem poles and spreads across the cortex. Before any visible changes in autofluorescence or browning, fragmented DNA was detected by TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling) in small clusters of inner cortical cells that subsequently could be distinguished cytologically from neighboring cells throughout rn root development. Inner cortical cells overlying lateral root primordia in either Rn or rn plants also were stained by TUNEL. Features commonly observed in animal cell apoptosis were confirmed by electron microscopy but, surprisingly, cells with a necrotic morphology were detected alongside apoptotic cells in the cortex of rn roots when TUNEL-positive cells were first observed. The two morphologies may represent different stages of a common pathway for programmed cell death (pcd) in plant roots, or two separate pathways of pcd could be involved. The phenotype of rn plants suggests that the Rn gene could either negatively regulate cortical cell death or be required for cortical cell survival. The possibility of a mechanistic link between cortical cell death in rn plants and during lateral root emergence is discussed.     

Localization of glycosaminoglycans and CD44 in the human lacrimal gland

Recent analyses of tears indicate the presence of glycosaminoglycans as their components, but their origin remains unknown. To further understand the origin of these tear components, we investigated by immunohistochemical techniques the localization of glycosaminoglycans and CD44 human lacrimal glands obtained from 20 cadavers at autopsy. Monoclonal antibodies to CD44, a receptor for hyaluronic acid, dermatan sulfate, chondroitin sulfate, and keratan sulfate were applied to the tissue. Hyaluronic acid binding region was also used for the staining of hyaluronic acid. By light microscopy, immunoreactivity for CD44 was mostly detected on the baso-lateral membrane of acinar and ductal cells, and the vascular endothelium in the interstitium. Positive staining of hyaluronic acid was associated intensely with the basal membrane of acinar and ductal cells and weakly, faintly or not at all with their lateral membrane. Positive staining of hyaluronic acid and immunoreactivity for dermatan sulfate were detected in interstitial fibrous structures; particularly, the former was intense in the perivascular fibrous structures, and the latter along the periparenchimal fibrous structures. Immunoreactivity for chondroitin sulfate and keratan sulfate was seen in some acinar cells and the acinar and ductal lumen. By electron microscopy, immunogold particles indication chondroitin sulfate sulfate or keratan sulfate labeled secretory granules of the acinar cells. Considering the fact that CD44 is a receptor molecule for hyaluronic acid, the association of hyaluronic acid with the basal membrane and weakly or faintly with the lateral membrane of acinar and ductal cells may be attributed to the expression of CD44 on the baso-lateral membrane of the cells. Moreover, the presence of immunoreactivity for chondroitin sulfate and keratan sulfate in secretory granules of acinar cells and their lumens suggests that tears from the lacrimal gland contain these glycosaminoglycans.     

S-layered Aneurinibacillus and Bacillus spp. are susceptible to the lytic action of Pseudomonas aeruginosa membrane vesicles

When S-layered strains of Bacillus stearothermophilus and Aneurinibacillus thermoaerophilus, possessing S-layers of different lattice type and lattice constant as well as S-(glyco)protein chemistry, and isogenic S-layerless variants were subjected to membrane vesicles (MVs) from P. aeruginosa during plaque assays on plates or CFU measurements on cell suspensions, all bacterial types lysed. Electron microscopy of negative stains, thin sections, and immunogold-labelled MV preparations revealed that the vesicles adhered to all bacterial surfaces, broke open, and digested the underlying peptidoglycan-containing cell wall of all cell types. Reassembled S-layer did not appear to be affected by MVs, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that the S-(glyco)proteins remained intact. meso-Diaminopimelic acid, as a peptidoglycan breakdown product, was found in all culture supernatants after MV attack These results suggest that even though MVs are much larger than the channels which penetrate these proteinaceous arrays, S-layers on gram-positive bacteria do not form a defensive barrier against the lytic action of MVs. The primary mode of attack is by the liberation from the MVs of a peptidoglycan hydrolase, which penetrates through the S-layer to digest the underlying peptidoglycan-containing cell wall. The S-layer is not affected by MV protease.     

Calorimetric and freeze fracture analysis of lipid phase transitions and lateral translational motion of intramembrane particles in mitochondrial membranes

Differential scanning calorimetry combined with freeze fracture electron microscopy reveals that thermotropic lipid phase transitions and lateral translational motion of intramembrane particles occur in both membranes of whole, intact rat liver mitochondria and in isolated inner and outer membranes. The onset temperature of the liquid crystalline to gel state lipid phase transition in whole mitochondria and in the isolated outer membrane fraction is biphasic with an initial transition exotherm occurring at 9 degrees C. The onset temperature of the transition exotherm of the isolated inner membrane occurs at -4 degrees C. The onset temperature of the lipid transition endotherm is -15 degrees C for whole mitochondria, the inner membrane, ane the outer membrane fractions. These calorimetric analyses reveal that the bilayer lipid in the inner, energy transducing membrane is more fluid than in the outer membrane. Mitochondrial membranes cooled to temperatures in the region of their transition exotherms and then frozen reveal striking lateral separations between smooth, intramembrane particle-free regions (rich in gel state lipid) and particle-dense regions (rich in integral proteins) in their hydrophobic fracture faces. Such thermotropic lipid-protein lateral separations are completely reversible. These freeze fracture observations suggest that both mitochondrial membranes are naturally fluid to the extent that the integrat membrane proteins can diffuse laterally in the bilayer lipid.