Motility of the vestibular hair cell of the guinea pig and bull frog

The motile response of the isolated vestibular hair cell induced by a neurotransmitter was studied. After application of both physostigmine and acetylcholine (Ach) as well as glutamic acid, shortening or tilting of the neck of the guinea pig hair cell was observed. These findings suggest that the effect of a neurotransmitter in the neck region as well as the efferent neuron is involved in the motile response. The location of F-action in isolated vestibular hair cells was investigated by using FITC-labeled phalloidin. In freeze-fixed vestibular hair cells, marked labeling was noted in the hair bundle, cuticular plate and throughout the cytoplasm. After application of both physostigmine and Ach, the labeling in the cuticular plate and the cytoplasm became more intense than that in the hair bundle. Alteration of this phalloidin-labeling pattern suggests that actin could play an important role in the self movement of vestibular sensory cells. The shape of the bull frog hair cell also changed after application of Ach. At the same time, spontaneous discharge and the time constant of the posterior semicircular canal nerve activity decreased. These results suggest that an adaptation mechanism induced by change in the cell shape and membrane potential inhibits the activity of the afferent neuron. Furthermore, these active events could be closely related to the active regulation of vestibular hair cell transmission.     

The consistent occurrence of a striated organelle (Friedmann body) in the inner hair cells of the normal chinchilla

Striated organelles have consistently been observed in electron micrographs of serial sections from the inner hair cells of normal chinchilla cochleas. The striated organelle is located in the infracuticular plate region. It lines the cuticular plate, and the direction, pattern and periodicity of the striations vary along its length. The striated organelle is seen in close association with the cell membrane, smooth endoplasmic reticulum, microtubules and mitochondria. The striated organelle may play an active role in inner hair cell function, and its proliferation under pathological conditions, as observed by others, may be accompanied by alterations in sensitivity of the inner hair cell to stimuli.     

Postnatal depression and social supports in Vietnamese, Arabic and Anglo-Celtic mothers

Nitric oxide synthase III (NOS III) was identified in the guinea pig cochlea on an ultrastructural level using a post-embedding immunolabeling procedure. Ultrathin sections of London Resin (LR) White-embedded specimens were incubated with various concentrations of a commercially available antibody to NOS III and the immunoreactivity visualized by a gold-labeled secondary antibody. Analysis of ultrathin sections of the organ of Corti in the second turn of the cochlea showed that NOS III could be localized in the endothelial cells of the blood vessels under the basilar membrane, which was comparable to its location in similar cells types in various biological systems. Besides this, NOS III was also found in the cytoplasm and in the nuclei of inner and outer hair cells. Immunoreactivity was not distributed homogeneously within receptor cells. Numerous gold particles could be identified at the border of the cuticular plates, in the middle parts of the stereocilia and in the cytoplasm. Gold-labeled anti-NOS III antibodies in these sites were seen mostly on the cytoplasmic side of the submembranous cisterns in the vicinity of mitochondria and in the central parts of the hair cells, whereas the cisterns were nearly free from any immunoreactivity. NOS III was also detected in the efferent and afferent nerve endings that were located at the basal and basolateral side of the outer hair cells. Some immunoreactivity was visible in different nerve fibers of the inner and outer spiral tunnels. Besides this, gold-labeled antibodies were also present in the cuticular plate of inner and outer pillar cells, in the cytoskeletal elements located in the apical parts of Deiters cells, forming the lamina reticularis, and in the cytoskeletal-containing region of the cytoplasm of those Deiters cells located at the basal side of the outer hair cells. The role of the NOS III immunoreactivity identified in the organ of Corti was consistent with respect to hair cell and tissue modulation.     

Studies on the posterior silk gland of the silkworm Bombyx mori. VI. Distribution of microtubules in the posterior silk gland cells

There are two microtubule systems in the posterior silk gland cells. One is a radial microtubule system in which the microtubules run radially from the basal to the apical cytoplasm and in which fibroin globules (secretory granules of fibroin) and mitochondria are arranged along these microtubules, thus composing a "canal system" which is assumed to be responsible for the intracellular transport of fibroin globules. The other is a circular microtubule system in the apical cytoplasm which is composed of bundles of microtubules and microfilaments running in a circular arrangement around the glandular lumen at an interval of approximately 4 mum at the end of the fifth instar. This system is presumably concerned with secretion and/or intraluminal transport of fibroin.     

Throat disorders

In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25-50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) approximately 40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.     

Motility of isolated vestibular hair cells induced by potassium

Many studies of the outer hair cells in cochlea have demonstrated active motility. However, very few studies have been done on vestibular hair cells. This study was designed to demonstrate the motile responses of isolated vestibular hair cells of the chick, induced by potassium promoting contraction. Reversible cell shape changes were observed in 4 of 6 type I hair cells and 3 of 5 type II hair cells by applying the contraction solution. The cell shape changes were revealed mainly in the cuticular plate and infracuticular region. It was suggested that contraction in the cuticular plate of the isolated hair cells might be converted into tension which increases the stiffness of the sensory hairs and restricts their motions, based on the results of the present study, and the structure of contractile proteins and hair behaviors reported by previous investigators.     

Immunocytochemical comparison of posttranslationally modified forms of tubulin in the vestibular end-organs of the gerbil: tyrosinated, acetylated and polyglutamylated tubulin

Specific antibodies against alpha-tubulin, acetylated alpha-tubulin, tyrosinated alpha-tubulin and polyglutamylated alpha- and beta-tubulin were used to compare the distribution of posttranslationally modified tubulin in the vestibular end-organs of the gerbil. Antibodies to acetylated tubulin labeled a dense network of microtubules in the hair cells and bundles of microtubule in the supporting cells. Nerve fibers within and below the epithelium were weakly labeled. This localization paralleled that seen with antibodies to alpha-tubulin which labeled all microtubules present in the cells. Antibodies to tyrosinated tubulin labeled networks and bundles of microtubules in both hair cells and supporting cells and in addition gave intense, diffuse labeling in the cytoplasm of both cell types. It also labeled the nerve fibers. Antibodies to polyglutamylated tubulin were localized mainly in nerve fibers, and in the calyces the labeled microtubules were found running circumferentially around the type I sensory hair cells. Thus, tyrosinated tubulin was found in the fine networks of microtubules in both the sensory and supporting cells. Acetylated tubulin was found in the dense networks and bundles of microtubules in the sensory and supporting cells, but did not colocalize with polyglutamylated tubulin, which was found predominantly in the nerve fibers. The labeling patterns for the tyrosinated tubulin and posttranslationally modified tubulins in the sensory and supporting cells of the vestibular end organs differ from that seen in the organ of Corti and may reflect differences in the stability of the microtubules and the mechanical properties of the sensory epithelium.     

Nursing seminar in Ulm: "I would take part again anytime"

Laser scanning confocal microscope (LSCM) enables one to observe both the surface structure and the inner configuration in the same specimen, by its possibility of direct, non-invasive serial optical sectioning of whole mounted specimens. The potential value of LSCM in the field of inner ear morphological study was evaluated. The configuration of upper parts of organ of Corti was observed with the LSCM combined with double-stained fluorescence immunohistochemistry technique. The actin filament of hair cells by phalloidin, and the cytokeratin of supporting cells by monoclonal pan-anticytokeratin antibody. The stereocilia, cuticular plate, and the cuticle-free area of hair cells were well demonstrated. In the same specimen, the head plate of outer pillar cell, the phalangeal apical plate and the phalangeal process of Deiter cells were clearly showed as well. LSCM provide a new tool to the morphological study of organ of Corti.     

VIP17/MAL, a proteolipid in apical transport vesicles

VIP17 is a proteolipid enriched in the CHAPS-insoluble complexes from MDCK cells, and a candidate component of the molecular machinery responsible for the sorting and targeting of proteins to the apical surface. Cloning and sequencing of the cDNA encoding the protein revealed that it is the canine homolog of the human and rat MAL proteins. Analysis by immunofluorescence microscopy of epitope-tagged VIP17/MAL expressed transiently in BHK cells and stably in MDCK cells revealed a perinuclear, vesicular, and plasmalemmal staining. In MDCK cells the distribution was mainly in vesicular structures in the apical cytoplasm. These and other results suggest that VIP17/MAL is an important component in vesicular trafficking cycling between the Golgi complex and the apical plasma membrane.     

The N-methyl-D-aspartate (NMDA) receptor is postsynaptic to substance P-containing axon terminals in the rat superficial dorsal horn

Cell differentiation often involves changes in cell polarity. In this study we show that neuroepithelial cells, the progenitors of all neurons and macroglial cells of the vertebrate central nervous system, downregulate the polarized delivery to the apical and basolateral plasma membrane domains during development. Upon infection of the neuroepithelium of mouse embryos with fowl plague virus (FPV), polarized delivery of the viral envelope hemagglutinin, an apical marker, occurred at the neural plate stage (E8), but was downregulated at the open neural tube stage (E9). Upon infection with vesicular stomatitis virus, the viral envelope G protein, a basolateral marker, showed an unpolarized delivery not only at the open neural tube stage, but already at the neural plate stage. These results show that a progressive downregulation of plasma membrane polarity of neuroepithelial cells precedes neural tube closure and the onset of neurogenesis.     

Partitioning of cytoplasmic organelles during mitosis with special reference to the Golgi complex

During mitosis, not only the genetic material stored in the nucleus but also the constituents of the cytoplasm should be equally partitioned between the daughter cells. For this sake, the dividing cell goes through an extensive structural reorganization and transport along the endocytic and exocytic pathways is temporarily arrested. Early in prophase, the radiating array of cytoplasmic microtubules disassembles and the membrane systems of the secretory apparatus start to split up. In metaphase, the nuclear envelope fragments and the condensing chromosomes associate with the forming mitotic spindle. The cisternal and tubular elements of the endoplasmic reticulum and the Golgi complex break down into small vesicles, presumably as the result of an imbalance between vesicle budding and fusion. In anaphase, the two sets of chromosomes are pulled apart and a cleavage furrow forms halfway between the spindle poles. Since most organelles occur in multiple and widely dispersed copies at this stage, they will be evenly distributed between the daughter cells. During telophase and cytokinesis, the preceding fragmentation process is reversed. A nuclear envelope reappears around the chromosomes and cytoplasmic microtubules reassemble. The endoplasmic reticulum is rebuilt as a continuous system of flattened cisternae and tubules. Stacks of Golgi cisternae arise from small vesicles and are rearranged in an interconnected network. In parallel, the biosynthetic functions of the cell are normalized and intracellular membrane traffic is resumed.     

Both microtubules and actin filaments are required for efficient postendocytotic traffic of the polymeric immunoglobulin receptor in polarized Madin-Darby canine kidney cells

It has been postulated that membrane traffic in polarized epithelial cells requires both actin filaments and microtubules. We have tested this hypothesis by analyzing the effect of cytochalasin D (cytoD; an actin-disrupting agent), by itself or in combination with nocodazole (a microtubule depolymerizing agent), on postendocytic traffic in Madin-Darby canine kidney cells. CytoD treatment inhibited basolateral to apical transcytosis of IgA in polymeric immunoglobulin receptor-expressing cells by approximately 45%, but had little effect on basolateral recycling of transferrin. Apical recycling of IgA was also inhibited by approximately 20%. Like nocodazole, cytoD acted at an early step in transcytosis, and inhibited translocation of IgA between the basolateral early endosomes and the apical recycling endosome. There was little inhibition of the subsequent release of IgA from the apical recycling endosome of cytoD- or nocodazole-treated cells. Order-of-addition experiments suggest that the cytoD-sensitive step preceded the nocodazole-sensitive step. Treatment with both cytoD and nocodazole inhibited transcytosis 95%. These results suggest that in addition to microtubules, efficient postendocytic traffic in polarized epithelial cells also requires actin filaments.     

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.     

Constitutive transport between the trans-Golgi network and the plasma membrane according to the maturation model. A hypothesis

Here we examine the application of the cisternal/carrier maturation model to describe transport of cargo proteins from the Golgi apparatus to the plasma membrane. Interpretation of the available evidence in the light of carrier maturation suggests that the transport intermediates between these stations are large pleiomorphic carriers formed by maturation of the trans-Golgi compartment, rather than vesicles, as would be postulated by the vesicular shuttle model. Mature carriers move along microtubules towards the plasma membrane via a microtubule/(kinesin)-based motor system. The maturation and vesicular transport models are compared in terms of consistency with the available literature.     

(Glyco)sphingolipids are sorted in sub-apical compartments in HepG2 cells: a role for non-Golgi-related intracellular sites in the polarized distribution of (glyco)sphingolipids

In polarized HepG2 cells, the fluorescent sphingolipid analogues of glucosylceramide (C6-NBD-GlcCer) and sphingomyelin (C6-NBD-SM) display a preferential localization at the apical and basolateral domain, respectively, which is expressed during apical to basolateral transcytosis of the lipids (van IJzendoorn, S.C.D., M.M. P. Zegers, J.W. Kok, and D. Hoekstra. 1997. J. Cell Biol. 137:347-457). In the present study we have identified a non-Golgi-related, sub-apical compartment (SAC), in which sorting of the lipids occurs. Thus, in the apical to basolateral transcytotic pathway both C6-NBD-GlcCer and C6-NBD-SM accumulate in SAC at 18 degreesC. At this temperature, transcytosing IgA also accumulates, and colocalizes with the lipids. Upon rewarming the cells to 37 degreesC, the lipids are transported from the SAC to their preferred membrane domain. Kinetic evidence is presented that shows in a direct manner that after leaving SAC, sphingomyelin disappears from the apical region of the cell, whereas GlcCer is transferred to the apical, bile canalicular membrane. The sorting event is very specific, as the GlcCer epimer C6-NBD-galactosylceramide, like C6-NBD-SM, is sorted in the SAC and directed to the basolateral surface. It is demonstrated that transport of the lipids to and from SAC is accomplished by a vesicular mechanism, and is in part microtubule dependent. Furthermore, the SAC in HepG2 bear analogy to the apical recycling compartments, previously described in MDCK cells. However, in contrast to the latter, the structural integrity of SAC does not depend on an intact microtubule system. Taken together, we have identified a non-Golgi-related compartment, acting as a "traffic center" in apical to basolateral trafficking and vice versa, and directing the polarized distribution of sphingolipids in hepatic cells.     

Functional morphology of insect mechanoreceptors

This paper reviews the structure and function of insect mechanoreceptors with respect to their cellular, subcellular, and cuticular organization. Four types are described and their function is discussed: 1, the bristles; 2, the trichobothria; 3, the campaniform sensilla; and 4, the scolopidia. Usually, bristles respond to touch, trichobothria to air currents and sound, campaniform sensilla to deformation of the cuticle, and scolopidia to stretch. Mechanoreceptors are composed of four cells: a bipolar sensory neuron, which is enveloped by the thecogen, the trichogen, and the tormogen cells. Apically, the neuron gives off a ciliary dendrite which is attached to the stimulus-transmitting cuticular structures. In types 1-3, the tip of the dendrite contains a highly organized cytoskeletal complex of microtubules, the "tubular body," which is connected to the dendritic membrane via short rods, the "membrane-integrated cones" (MICs). The dendritic membrane is attached to the cuticle via fine attachment fibers. The hair-type sensilla (types 1, 2) are constructed as first-order levers, which transmit deflection of the hair directly to the dendrite tip. In campaniform sensilla (type 3), there is a cuticular dome instead of a hair and the dendrite is stimulated by deformation of the cuticle. In these three types, a slight lateral compression of the dendrite tip is most probably the effective stimulus. In scolopidia, the dendritic membrane is most probably stimulated by stretch. On the subcellular level, connectors between the cytoskeleton, the dendritic membrane, and extracellular (cuticular) structures are present in all four types and are thought to be engaged in membrane depolarization.     

Fodrin is a constituent of the cortical lattice in outer hair cells of the guinea pig cochlea: immunocytochemical evidence

Localization of fodrin, a membrane skeletal protein, in the outer hair cell of the guinea pig cochlea was examined by immunocytochemical techniques. By immunofluorescence microscopy, fodrin was observed in the cuticular plate, in the infracuticular network and along the lateral wall. By immunoelectron microscopy of ultrathin cryosections, labeling for fodrin along the lateral wall was localized between the cell membrane and the outermost layer of the subsurface cisternae. Furthermore, pre-embedding immunoelectron microscopy of permeabilized specimens showed that most immunogolds for fodrin were on the thin cross-linking component of the cortical lattice. The results indicate that fodrin is a constituent of the cortical lattice which is thought to play an important role in outer hair cell motility.     

The cuticular plate of the hair cell in relation to morphological gradients of the chicken basilar papilla

The aim of the present study was to provide details on the diversity and morphological gradients in the anatomy of the cuticular plate of hair cells in the chicken basilar papilla. The structure of the cuticular plate, which is mainly made up of a network of actin filaments, may be related to differences in the mechanical demands on the anchorage of the stereovillar bundle. We describe the morphological gradients in the cuticular plates as seen in transverse section for four positions along the basilar papilla. Three different shapes of cuticular plate could be distinguished. In general, cuticular plates in neurally-lying hair cells have their main mass on the neural side of the cells; for abneural cells, the converse is true. The shape of the plates changes gradually across the papilla; symmetrical forms exist. The hair-cell bundle orientation (and thus the preferred direction of stimulation of the bundle), as measured using scanning EM preparations, does not correlate with the shape of the plate in transverse section. The present data confirm the notion developed from other studies that (1) there are no distinct populations of hair cells, (2) there are no linear or monotonic morphological gradients, and (3) the gradients on the papilla are species- and position-specific.     

Association of Rab25 and Rab11a with the apical recycling system of polarized Madin-Darby canine kidney cells

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin-Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.     

Branch migration through DNA sequence heterology

We have studied the biosynthesis and transport of the endogenous caveolins in MDCK cells. We show that in addition to homooligomers of caveolin-1, heterooligomeric complexes of caveolin-1 and -2 are formed in the ER. The oligomers become larger, increasingly detergent insoluble, and phosphorylated on caveolin-2 during transport to the cell surface. In the TGN caveolin-1/-2 heterooligomers are sorted into basolateral vesicles, whereas larger caveolin-1 homooligomers are targeted to the apical side. Caveolin-1 is present on both the apical and basolateral plasma membrane, whereas caveolin-2 is enriched on the basolateral surface where caveolae are present. This suggests that caveolin-1 and -2 heterooligomers are involved in caveolar biogenesis in the basolateral plasma membrane. Anti-caveolin-1 antibodies inhibit the apical delivery of influenza virus hemagglutinin without affecting basolateral transport of vesicular stomatitis virus G protein. Thus, we suggest that caveolin-1 homooligomers play a role in apical transport.