Lanthanides inhibit the human noradrenaline, 5-hydroxytryptamine and dopamine transporters

The spermatozoon of the monopisthocotylean monogenean Pseudodactylogyrus sp. (a gill parasite of eels) has a single axoneme showing a 9 + '1' pattern, a nucleus and a mitochondrion, but has no cortical microtubules. This species thus provides a very simple model for the study of tubulin in the 9 + '1' axonemes of the Platyhelminthes, in contrast with digenean sperm which have a more complex spermatozoon with two such axonemes and cortical microtubules. Indirect immunofluorescence labelling of tubulin shows that the elongating spermatids, initially lying in all directions in the early stages, are arranged as parallel elements in further stages. The number of spermatids in an isogenic group could also be precisely counted and equals 32. Nuclear labelling with fluorescent dyes shows that the nuclei, first located in the common mass of the spermatids, later elongate and migrate into the growing spermatids, and that the nucleus is located in the central part of the mature spermatozoon, with the two extremities devoid of nucleus. Labelling with antibodies directed against acetylated, tyrosinated, and polyglutamylated tubulin gave positive results, thus indicating that these post-translational modifications of tubulin are present in the axoneme of spermatids and spermatozoa of monopisthocotylean monogeneans.     

Intracellular distributions and putative functions of calcium-binding proteins in the bullfrog vestibular otolith organs

Hair cells in the bullfrog vestibular otolith organs were immunolabeled by monoclonal and polyclonal antisera against calbindin (CaB), calmodulin (CaM), calretinin (CaR), and parvalbumin (PA). S-100, previously shown to immunolabel striolar hair cells in fish vestibular organs, only weakly immunolabeled hair cells in the bullfrog vestibular otolith organs. Immunolabeling was not detected in supporting cells. With the exception of CaR, myelinated axons and unmyelinated nerve terminals were immunolabeled by all of the above antisera. Immunolabeling was seen in all saccular hair cells, although hair cells at the macular margins were immunolabeled more intensely for CaB, CaM, and PA than more centrally located hair cells. As the macula margins are known to be a growth zone, this labeling pattern suggests that marginal hair cells up-regulate their calcium-binding proteins during hair cell development. In the utriculus, immunolabeling for CaM and PA was generally restricted to striolar hair cells. CaR immunolabeling was restricted to the stereociliary array. Immunolabeling for other calcium-binding proteins was generally seen in both the cell body and hair bundles of hair cells, although this labeling was often localized to the stereociliary array and the apical portion of the cell body. CaM and PA immunolabeling in the stereociliary array in saccular and utricular striolar cells suggests a functional role for these proteins in mechanoelectric transduction and adaptation.     

Innervation of cholinergic vestibular efferent system in vestibular periphery of rats

The innervation of cholinergic efferent fibers in the vestibular endorgans of the rats was investigated using a modified preembedding immunostaining technique of immunoelectron microscopy. A monoclonal antibody to choline acetyltransferase (ChAT) was used as a marker of cholinergic fibers. It was found that there were four types of cholinergic innervation in the vestibular endorgans of the rat: (1) cholinergic nerve endings formed axo-dendritic synapses with afferent chalice surrounding the type I sensory hair cells; (2) cholinergic nerve endings formed axo-somatic synapses with type II hair cells; (3) cholinergic fibers synapse with afferent nerve fibers and (4) a synaptic contact developed between cholinergic nerve endings. The results demonstrated that a multiform innervation of the cholinergic efferents exists in the rats vestibular periphery.     

Mapping surface sequences of the tubulin dimer and taxol-induced microtubules with limited proteolysis

Native tubulin alpha beta dimers and microtubules have been subjected to limited proteolysis with trypsin, chymotrypsin, elastase, clostripain, proteinase lysine-C, thermolysin, protease V8, papain, subtilisin, proteinase K, proteinase aspartic-N, and bromelain. Eighty nicking points have been mapped onto the alpha- and beta-tubulin sequences with the aid of site-directed antibodies, of which 18 sites have been exactly determined by N-terminal sequencing, and the probable position of 6 others deduced from protease specificities. Proteolytic sites cluster into five characteristic zones, including the C termini of both chains. Residues accessible to proteases in the tubulin dimer include alpha-tubulin Lys40-Thr41-Ile42, Glu168-Phe169-Ser170, Ser178-Thr179-Ala180-Val181, Lys280-Ala281, Glu290-Ile291, Ala294-Cys295, Arg339-Ser340 (plus probably Lys60-His61 and Glu183-Pro184) and beta-tubulin Gly93-Gln94, Lys174-Val175, Gly277-Ser278, Tyr281-Arg282-Ala283, Cys354-Asp355 (plus probably Arg121-Lys122, Phe167-Ser168, Tyr183-Asn184, and Glu426-Asp427 or Ala430-Asp431). While the majority of these sites remain accessible at the outer surface of taxol-induced microtubules, alpha-tubulin Lys280-Ala281, Arg339-Ser340 and beta-tubulin Tyr281-Arg282-Ala283 (and probably Arg121-Lys122) become protected from limited proteolysis, suggesting that they are close to or at intermolecular contacts in the assembled structure. The protease nicking points constitute sets of surface constraints for any three-dimensional model structures of tubulin and microtubules. The dimer tryptic site at alpha-tubulin 339-340 jumps approximately 12-22 residues upstream (probably to Lys326-Asp327 or Lys311-Tyr312) in taxol microtubules, suggesting a tertiary structural change. The cleavage of the approximately 10 C-terminal residues of alpha-tubulin by protease V8, papain, and subtilisin is inhibited in taxol microtubules compared to tubulin dimers, while the approximately 20 C-terminal residues of beta-tubulin are similarly accessible to protease V8, subtilisin, proteinase K, proteinase AspN, and bromelain and show enhanced papain cleavage. This is consistent with models in which the alpha-tubulin C-terminal zone is near the interdimer contact zone along the protofilaments, whereas the C terminus of beta is near the interface between both subunits.     

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.     

Organization of choline acetyltransferase-containing structures in the cranial nerve motor nuclei and spinal cord of the monkey

In the vertebrate vestibular periphery, gamma-aminobutyric acid (GABA) has long been presumed to be a neurotransmitter candidate. However, experimental reports about the localization and function of GABA in the vestibular systems of vertebrates are contradictory. In addition, there is no information in the literature concerning the localization of GABA in the human vestibular periphery. The present study investigates the ultrastructural localization of GABA-like immunoreactivity in the human utricular macula. A modified pre-embedding immunostaining electron microscopy technique was applied using two different commercially available polyclonal antibodies to GABA. GABA-like immunoreactivity is confined to the vesiculated nerve fibers and terminals of the human vestibular neurosensory epithelia. The GABA-containing nerve terminals make asymmetrical axo-dendritic synapses with the afferent chalices surrounding the type I sensory hair cells. Type I and type II hair cells as well as afferent chalices are devoid of GABA-like immunoreactive staining. The present study demonstrates that GABA exists in the human vestibular periphery, and that GABA is a neurotransmitter candidate of the human efferent vestibular system.     

Tubulin polyglycylation in Platyhelminthes: diversity among stable microtubule networks and very late occurrence during spermiogenesis

The distribution of glycylated tubulin has been analyzed in different populations of stable microtubules in a digenean flatworm, Echinostoma caproni (Platyhelminthes). Two cellular types, spermatozoa and ciliated excretory cells, have been analyzed by means of immunofluorescence, immunogold, and immunoblotting techniques using two monoclonal antibodies (mAbs), AXO 49, and TAP 952, specifically directed against differently glycylated isoforms of tubulin. The presence of glycylated tubulin in the two cell types was shown. However, the differential reactivities of TAP 952 and AXO 49 mAbs with the two axoneme types suggest a difference in their glycylation level. In addition, within a single cell, the spermatozoon, cortical microtubules underlying the flagellar membrane, and axonemal microtubules were shown to comprise different tubulin isoforms, the latter ones only being labelled with one of the antiglycylated tubulin mAbs, TAP 952. Similarly, the antiacetylated (6-11B-1) and polyglutamylated (GT335) tubulin mAbs decorated the two types of axonemal microtubules, but not the cortical ones. From these data, a subcellular sorting of posttranslationally modified tubulin isoforms within spermatozoa, on the one hand, and a cellular sorting of glycylated isoforms inside the whole organism, on the other hand, is demonstrated in the flatworm E. caproni. Last, a sequential occurrence of tubulin posttranslational modifications was observed in the course of spermiogenesis. Acetylation appears first, followed shortly by glutamylation; glycylation takes place at the extreme end of spermiogenesis and, specifically, in a proximo-distal process. Thus in agreement with, and extending other studies [Bré et al., 1996], glycylation appears to close the sequence of posttranslational events occurring in axonemal microtubules during spermiogenesis.     

Arrangement of vestibular nerve fibers in the semicircular canal crista of the chinchilla

The topographic arrangement of vestibular nerve fibers innervating semicircular canal cristae of the chinchilla was studied using computer-aided video-microscopy and three-dimensional reconstruction. At the level 20 microns proximal to the base of the crista, bundles consisting of 30-50 nerve fibers each were identified. Nerve fibers in bundles were classified into seven categories depending on the diameter. We confirmed that large nerve fibers were more frequently found in the central bundles and small nerve fibers were more frequently found in the peripheral bundles. The central bundle might function as a physiological unit coding various types of head movements, whereas the peripheral bundle might contribute more to the detection of slow and long-lasting movements giving rise to tonus and posture changes. The canalicular nerve may code rotational acceleration of the head via function- and locus-specific nerve fiber bundles.     

Hair cell recovery in the vestibular sensory epithelia of mature guinea pigs

The progression of recovery of the vestibular sensory epithelia of guinea pigs after gentamicin-induced hair cell injury was assessed quantitatively and qualitatively. Evaluations were made of the number of cells bearing hair bundles by using scanning electron microscopy (SEM) and of identifiable hair cells in thin sections. Both assessment procedures showed that an initial loss of hair cells in utricular maculae is followed by significant recovery in the number of hair cells present. SEM also showed recovery in saccules comparable to that in utricles. During the recovery, progressive maturation of hair bundles, which exhibited features similar to those seen during normal ontogenetic development of hair cells, could be identified. The pattern and extent of hair cell loss and subsequent reappearance revealed by SEM corresponded with that derived from analysis of thin sections. This suggests that repair of nonlethally damaged hair cells is unlikely but, rather, that new hair cells are produced. An apparent decrease in supporting cell numbers was observed coincident with the increase in hair cell numbers. This complements previous morphological observations, which have suggested new hair cells arise from direct, nonmitotic transdifferentiation of supporting cells. The quantitative analyses indicate that more than half of the hair cells that are lost are replaced, but the recovery process does not result in complete restoration of the epithelium. Eight months after the end of drug treatment, the number of hair cells present was still significantly less than normal, and several other abnormalities persisted. There was also no evidence of any hair cell recovery in the organ of Corti. Thus, there appear to be limitations on the capacity for spontaneous replacement of lost hair cells in the mammalian inner ear.     

A role for BDNF in early postnatal rat vestibular epithelia maturation: implication of supporting cells

The early development of the inner ear is largely determined by two members of the neurotrophic family: brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT-3). Little information is available on the role of these neurotrophins during the late stages of vestibular development in the rat which take place during the first postnatal weeks. At this period where terminal synaptogenesis and maturation occur, we have investigated the expression and the activity of BDNF, the most important neurotrophin in the vestibular system. Using different experimental approaches, we show that BDNF is released by vestibular epithelia on postnatal day 3 (P3) and continues to have a trophic effect on vestibular neurones in vitro. Immunocytochemistry coupled to confocal microscopy revealed a remarkable evolution in BDNF localization during later stages of development. Whereas BDNF is present in both supporting cells and hair cells at P3, its distribution gradually changed and is highly compartmentalized within the upper part of supporting cells at P8 and P15. In parallel, we observed the presence of a truncated form of the BDNF receptor in sensory hair cells. These results suggest an original role for supporting cells, which could be involved in the release of BDNF during the late stages of synaptogenesis in mammalian vestibular epithelia. In particular, BDNF could participate to the set up of the calyx, a specific nerve structure surrounding type I vestibular hair cells.     

Separation of tubulin subunits under nondenaturing conditions

The dissociation and separation of the tubulin alpha- and beta-subunits have been achieved by binding alpha-subunits to an immunoadsorbent gel and selectively inducing release of free beta-subunits. The immunoadsorbent gel was prepared by coupling the monoclonal antibody YL1/2 to Sepharose 4B which specifically recognizes the C-terminal end of tyrosinated alpha-subunits. Extensive tubulin subunit dissociation and separation occurred in Tris buffer at neutral pH but was greatly enhanced at basic pHs (8. 0-8.5). The binding of colchicine to heterodimeric tubulin resulted in a marked protection against dissociation. The dissociation of tubulin subunits was accompanied by loss of colchicine binding capacity, and ability to polymerize into microtubules. As shown by circular dichroism, loss of functional properties was not due to extensive denaturation of tubulin, as tubulin retained most of its secondary structure. Neither of the separated alpha- or beta-subunits was able to bind colchicine, but functional tubulin that was able to bind colchicine could be reconstituted from the dissociated subunits by changing the buffer to a neutral mixture of Tris and Pipes. The yield of reconstitution, as estimated from kinetic measurements of colchicine binding capacity, amounted to about 25%. Such a yield can probably be improved with minor changes in experimental conditions. The quantitative dissociation of tubulin into separated "native" alpha- and beta-subunits should provide a powerful tool for further studies on the properties of the individual tubulin subunits and the structure-function relationships of the tubulins.     

Differential glycosylation of auditory and vestibular hair bundle proteins revealed by peanut agglutinin

Up to four morphologically distinct types of cross-link are found between the stereocilia in the hair bundles of avian hair cells. These links are involved in mechanotransduction, force transmission across the bundle, and maintenance of hair bundle structure. They appear to be specialisations of the cell coat, but very little is known about their molecular composition. Chick inner ear tissues were therefore screened with a number of different lectins to find markers for specialisations of the hair bundle surface. One lectin, peanut agglutinin (PNA), which recognises the dissacharide Gal beta 1-3GalNAc, was found to be a fairly selective marker for vestibular hair bundles, but it does not stain the stereocilia of auditory hair cells. The staining patterns observed with PNA in the vestibular system closely resemble those seen with a monoclonal antibody (mab) directed against a 275 kD component of the hair cell's apical surface known as the hair-cell antigen (HCA). However, unlike PNA, the mab recognises both vestibular and auditory hair cells. A detailed comparison of the fluorescence staining patterns observed with PNA and the anti-HCA mab indicates that binding sites for both ligands spatially codistribute on the surface of vestibular hair cells. The lectin and the anti-HCA mab binding sites are both sensitive to trypsin treatment, and, with sections of the vestibular system, PNA pretreatment blocks subsequent anti-HCA mab staining. Immunoelectron microscopy of vestibular hair bundles shows that PNA and the anti-HCA mab both label a type of cross-link known as the shaft connector. This link type is present on both auditory and vestibular hair bundles but reacts with PNA only in the vestibular system. The lectin jacalin, which has greater specificity for Gal beta 1-3GalNAc than does PNA, also only labels vestibular and not auditory hair bundles. Although terminal sialic acid residues can block both PNA and jacalin binding, neuraminidase treatment does not unmask cryptic binding sites for these lectins on auditory hair cells but does reveal PNA and jacalin staining at a number of other locations in the inner ear. The results obtained with the lectins PNA and jacalin indicate that either the HCA or other components of the shaft links are differentially glycosylated in the vestibular and auditory epithelia of the bird. The functional significance for such a difference in glycosylation remains to be determined, but auditory and vestibular hair cells operate over different frequency ranges, and variations in glycosylation might confer different micromechanical properties on the hair bundles in these two systems.     

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.     

S100A1 regulates neurite organization, tubulin levels, and proliferation in PC12 cells

As a first step in determining what cellular processes are regulated by the calcium-modulated protein S100A1 isoform in neurons, the effects of ablated S100A1 expression on neurite organization and microtubule/tubulin levels in PC12 cells were examined. A mammalian expression vector containing the rat S100A1 cDNA in the antisense orientation with respect to a cytomegalovirus promoter was constructed and transfected into PC12 cells. Indirect immunofluorescence microscopy confirmed decreased S100A1 protein levels in all three stable transfectants (pAntisense clones) that expressed exogenous S100A1 antisense mRNA. In response to nerve growth factor, pAntisense clones extended significantly more neurites than control cells (4.01 +/- 0.16 versus 2.93 +/- 0.16 neurites/cell). This increase in neurite number was accompanied by an increase in total alpha-tubulin levels in untreated (4.0 +/- 0.6 versus 1.76 +/- 0.4 ng of alpha-tubulin/mg of total protein) and nerve growth factor-treated pAntisense clones (4.15 +/- 0.4 versus 2. 04 +/- 0.5 ng of alpha-tubulin/mg of total protein) when compared with control cells. At high cell densities, pAntisense clones exhibited a significant decrease in anchorage-dependent growth. In soft agar, pAntisense clones formed significantly more colonies (153 +/- 8%) than control cells (116 +/- 5%). However, the pAntisense soft agar colonies were significantly smaller than those observed in control cells (40.6 +/- 3.0 versus 59.5 +/- 1.2 micron). These data suggest that cell density inhibits both anchorage-independent and -dependent growth of pAntisense clones. In summary, ablation of S100A1 expression in PC12 cells results in increased tubulin levels, altered neurite organization, and decreased cell growth. Thus, S100A1 may directly link the cytoskeleton and calcium signal transduction pathways to cell proliferation.     

The microtubule-stabilizing agent discodermolide competitively inhibits the binding of paclitaxel (Taxol) to tubulin polymers, enhances tubulin nucleation reactions more potently than paclitaxel, and inhibits the growth of paclitaxel-resistant cells

The lactone-bearing polyhydroxylated alkatetraene (+)-discodermolide, which was isolated from the sponge Discodermia dissoluta, induces the polymerization of purified tubulin with and without microtubule-associated proteins or GTP, and the polymers formed are stable to cold and calcium. These effects are similar to those of paclitaxel (Taxol), but discodermolide is more potent. We confirmed that these properties represent hypernucleation phenomena; we obtained lower tubulin critical concentrations and shorter polymers with discodermolide than paclitaxel under a variety of reaction conditions. Furthermore, we demonstrated that discodermolide is a competitive inhibitor with [3H]paclitaxel in binding to tubulin polymer, with an apparent Ki value of 0.4 microM. Multidrug-resistant human colon and ovarian carcinoma cells overexpressing P-glycoprotein, which are 900- and 2800-fold resistant to paclitaxel, respectively, relative to the parental lines, retained significant sensitivity to discodermolide (25- and 89-fold more resistant relative to the parental lines). Ovarian carcinoma cells that are 20-30-fold more resistant to paclitaxel than the parental line on the basis of expression of altered beta-tubulin polypeptides retained nearly complete sensitivity to discodermolide. The effects of discodermolide on the reorganization of the microtubules of Potorous tridactylis kidney epithelial cells were examined at different times. Intracellular microtubules were reorganized into bundles in interphase cells much more rapidly after discodermolide treatment compared with paclitaxel treatment. A variety of spindle aberrations were observed after treatment with both drugs. The proportions of the different types of aberration were different for the two drugs and changed with the length of drug treatment.     

Sound damage and gentamicin treatment produce different patterns of damage to the efferent innervation of the chick cochlea

Both sound exposure and gentamicin treatment cause damage to sensory hair cells in the peripheral chick auditory organ, the basilar papilla. This induces a regeneration response which replaces hair cells and restores auditory function. Since functional recovery requires the re-establishment of connections between regenerated hair cells and the central nervous system, we have investigated the effects of sound damage and gentamicin treatment on the neuronal elements within the cochlea. Whole-mount preparations of basilar papillae were labeled with phalloidin to label the actin cytoskeleton and antibodies to neurofilaments, choline acetyltransferase, and synapsin to label neurons; and examined by confocal laser scanning microscopy. When chicks are treated with gentamicin or exposed to acoustic overstimulation, the transverse nerve fibers show no changes from normal cochleae assayed in parallel. Efferent nerve terminals, however, disappear from areas depleted of hair cells following acoustic trauma. In contrast, efferent nerve endings are still present in the areas of hair cell loss following gentamicin treatment, although their morphological appearance is greatly altered. These differences in the response of efferent nerve terminals to sound exposure versus gentamicin treatment may account, at least in part, for the discrepancies reported in the time of recovery of auditory function.     

Centriole disassembly in vivo and its effect on centrosome structure and function in vertebrate cells

Glutamylation is the major posttranslational modification of neuronal and axonemal tubulin and is restricted predominantly to centrioles in nonneuronal cells (Bobinnec, Y., M. Moudjou, J.P. Fouquet, E. Desbruyères, B. Eddé, and M. Bornens. 1998. Cell Motil. Cytoskel. 39:223-232). To investigate a possible relationship between the exceptional stability of centriole microtubules and the compartmentalization of glutamylated isoforms, we loaded HeLa cells with the monoclonal antibody GT335, which specifically reacts with polyglutamylated tubulin. The total disappearance of the centriole pair was observed after 12 h, as judged both by immunofluorescence labeling with specific antibodies and electron microscopic observation of cells after complete thick serial sectioning. Strikingly, we also observed a scattering of the pericentriolar material (PCM) within the cytoplasm and a parallel disappearance of the centrosome as a defined organelle. However, centriole disappearance was transient, as centrioles and discrete centrosomes ultimately reappeared in the cell population. During the acentriolar period, a large proportion of monopolar half-spindles or of bipolar spindles with abnormal distribution of PCM and NuMA were observed. However, as judged by a quasinormal increase in cell number, these cells likely were not blocked in mitosis. Our results suggest that a posttranslational modification of tubulin is critical for long-term stability of centriolar microtubules. They further demonstrate that in animal cells, centrioles are instrumental in organizing centrosomal components into a structurally stable organelle.     

Hair cell formation in cultures of dissociated cells from the vestibular sensory epithelium of the bullfrog

HYPOTHESIS: Bullfrog vestibular hair cells are capable of regenerating in vitro. BACKGROUND: Recent studies have established that sensory organs in the inner ear of vertebrates continue to produce hair cells after birth. However, the mechanisms responsible for the regulation of this process are not well understood. The current study reports the development of a novel method for the culture of dispersed cells from the bullfrog inner ear. METHODS: New hair cell formation in this in vitro preparation was shown by sequential photomicroscopy. Studies with the selective marker for mitotic activity 5-bromo-2-deoxyuridine (BrdU) were done to estimate the level of cell proliferation and to quantify postmitotic hair cell formation. Finally, confirmation of cell type was obtained by scanning electron microscopy and by the use of specific markers for hair cells. RESULTS: Once the optimal culture conditions were established in the initial experiments, the formation of new hair cells was directly visualized in all unstained live cultures and fixed preparations without exception. Asymmetric division of progenitor cells, with subsequent differentiation of one of the daughter cells into new hair cells, also was documented by photomicroscopy. Approximately 12% of the cells were labeled with BrdU, of which 6% were hair cells, showing that new hair cell formation was subsequent to mitotic division in vitro. The identity of newly formed hair cells was verified as follows: 1) morphologically by scanning electron microscopy; 2) by positive labeling with phalloidin-rhodamine, a marker for actin; and 3) by positive calmodulin immunocytochemistry. CONCLUSIONS: This study reports the development of an in vitro culture preparation in which undifferentiated epithelial cells proliferate to become new hair cells. Evidence is provided of division of hair cell progenitors and subsequent differentiation of the daughter cells as one of the mechanisms involved in new hair cell formation in the culture preparation. This newly developed cell culture technique provides a powerful tool for further study of the process of hair cell formation in the vestibular end organ.     

Glycolytic enzymes and assembly of microtubule networks

The ability of glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase (PK), and lactate dehydrogenase muscle-type (LDH(M)), to generate interactive microtubule networks was investigated. Bundles have previously been defined as the parallel alignment of several microtubules and are one form of microtubule networks. Utilizing transmission electron microscopy, interactive networks of microtubules as well as bundles were readily observed in the presence of GAPDH, aldolase, or PK. These networks appear morphologically as cross-linked microtubules, oriented in many different ways. Light scattering indicated that the muscle forms of GAPDH, aldolase, PK and LDH(m) caused formation of the microtubule networks. Triose phosphate isomerase (TPI) and lactate dehydrogenase heart-type (LDH(H)), glycolytic enzymes which do not interact with tubulin or microtubules, did not produce bundles, or interactive networks. Sedimentation experiments confirmed that the enzymes that cross-link also co-pellet with the microtubules. Such cross-linking of microtubules indicate that the enzymes are multivalent with the capability of simultaneous binding to more than one microtubule.