Die mechanochemische Kopplung in der Hörzelle benötigt ein intaktes Zytoskelett

Summary Monospecific antibodies to actin and to tubulin were used as immunofluorescent and ferritin-labelled probes to evaluate the distribution of microtubules and actin filaments in the hearing organs from mouse, guinea pig and fish. The results indicate that in cochlear receptor cells actin and actin filaments as well as tubulin and microtubules are integral cytoskeletal elements. The presence of actin suggests a possible contractile mechanism within the sensory cilia whereas tubulin is thought to play an important role in the stability of sensory cells. Both proteins are discussed to form structural elements required for the mechano-chemical coupling in hearing.

---

---

Ausführliche Publikation in Arch Otorhinolaryngol (1981) 230: 81     

Actin-binding and microtubule-associated proteins in the organ of Corti.

Actin-binding and microtubule-associated proteins regulate microfilament and microtubule number, length, organization and location in cells. In freeze-dried preparations of the guinea pig cochlea, both actin and tubulin are found in the sensory and supporting cells of the organ of Corti. Fodrin (brain spectrin) co-localized with actin in the cuticular plates of both inner and outer hair cells and along the lateral wall of the outer hair cells. Alpha-actinin co-localized with actin in the cuticular plates of the hair cells and in the head and foot plates of the supporting cells. It was also found in the junctional regions between hair cells and supporting cells. Profilin co-localized with actin in the cuticular plates of the sensory hair cells. Myosin was detected only in the cuticular plates of the outer hair cells and in the supporting cells in the region facing endolymph. Gelsolin was found in the region of the nerve fibers. Tubulin is found in microtubules in all cells of the organ of Corti. In supporting cells, microtubules are bundled together with actin microfilaments and tropomyosin, as well as being present as individual microtubules arranged in networks. An intensely stained network of microtubules is found in both outer and inner sensory hair cells. The microtubules in the outer hair cells appear to course throughout the entire length of the cells, and based on their staining with antibodies to the tyrosinated form of tubulin they appear to be more dynamic structures than the microtubules in the supporting cells. The microtubule-associated protein MAP-2 is present only in outer hair cells within the organ of Corti and co-localizes with tubulin in these cells. No other MAPs (1,3,4,5) are present. Tau is found in the nerve fibers below both inner and outer hair cells and in the osseous spiral lamina. It is clear that the actin-binding and microtubule-associated proteins present in the cochlea co-localize with actin and tubulin and that they modulate microfilament and microtubule structure and function in a manner similar to that seen in other cell types. The location of some of these proteins in outer hair cells suggests a role for microfilaments and microtubules in outer hair cell motility.     

Actin filaments and microtubules regulate endocytosis in marginal cells of the stria vascularis

Conclusion. Cationized ferritin (CF) was internalized via clathrin-mediated endocytosis. This process depends on clathrin, actin filaments, and microtubules. Microperoxidase (MPO) was internalized via a clathrin- and caveolin-independent endocytic pathway, which was partially dependent on microtubules but independent of clathrin and actin filaments. Objective. We investigated the role of actin filaments and microtubules in the transport of endocytic carrier vesicles (ECVs) from the plasma membrane to the early sorting endosomes, using CF and MPO as tracers. Materials and methods. Fifty-five guinea pigs were used. The animals were divided into a CF endocytosis group and an MPO endocytosis group. These groups consisted of control, nocodazole-treated, cytochalasin (Cyt D)-treated, Cyt D + nocodazole-treated, and geldanamycin-treated subgroups. Results. For CF endocytosis, the following results were obtained. In the nocodazole experiment, in which microtubules were disrupted to form monomeric tubulin, the number of ECVs loaded with CF was greatly decreased. In the Cyt D experiment, in which the actin filaments were disrupted to form monomers, the number of ECVs labeled with CF was also greatly decreased. In the geldanamycin experiment, in which clathrin-mediated endocytosis was regulated and actin stress fibers were dissolved, the endocytosis of CF was severely inhibited. For MPO endocytosis, in the nocodazole experiment, the endocytosis of MPO was markedly suppressed.     

Immunohistochemical localization of several cytoskeletal proteins in inner ear sensory and supporting cells

Several structural and contractile proteins have been searched for with immunohistochemical methods using antibodies directed against these proteins. Three types of preparations from the guinea pig have been used: isolated stereocilia from the utricle, organ of Corti fragments obtained by cellular dissociation and 0.2-1 micrometer sections obtained by cryoultramicrotomy. The main finding is that different sets of proteins compose the cytoskeleton in supporting cells and the mechanoreceptor structures of the sensory cells. Thus, actin was found in association with fimbrin in the mechanoreceptive region of hair cells, whereas supporting cells, although rich in actin, did not reveal fimbrin. Instead tubulin was seen together with actin in supporting cells which also exhibited prekeratin. Fimbrin appears to function as a protein capable of making bundles and networks from actin filaments. Its exclusive presence in the mechanosensitive region of the sensory cells is possibly related to the function of these cells as mechanoreceptors.     

Actin filaments and microtubules regulate endocytosis in marginal cells of the stria vascularis

CONCLUSION: Cationized ferritin (CF) was internalized via clathrin-mediated endocytosis. This process depends on clathrin, actin filaments, and microtubules. Microperoxidase (MPO) was internalized via a clathrin- and caveolin-independent endocytic pathway, which was partially dependent on microtubules but independent of clathrin and actin filaments. OBJECTIVE: We investigated the role of actin filaments and microtubules in the transport of endocytic carrier vesicles (ECVs) from the plasma membrane to the early sorting endosomes, using CF and MPO as tracers. MATERIALS AND METHODS: Fifty-five guinea pigs were used. The animals were divided into a CF endocytosis group and an MPO endocytosis group. These groups consisted of control, nocodazole-treated, cytochalasin (Cyt D)-treated, Cyt D + nocodazole-treated, and geldanamycin-treated subgroups. RESULTS: For CF endocytosis, the following results were obtained. In the nocodazole experiment, in which microtubules were disrupted to form monomeric tubulin, the number of ECVs loaded with CF was greatly decreased. In the Cyt D experiment, in which the actin filaments were disrupted to form monomers, the number of ECVs labeled with CF was also greatly decreased. In the geldanamycin experiment, in which clathrin-mediated endocytosis was regulated and actin stress fibers were dissolved, the endocytosis of CF was severely inhibited. For MPO endocytosis, in the nocodazole experiment, the endocytosis of MPO was markedly suppressed.     

Cytoskeletal organization in the supporting cell of the guinea pig organ of Corti

The rapid-freeze, deep-etch method was used to visualize the three-dimensional organization of cytoskeletons in the supporting cells of the guinea pig organ of Corti. Deep-etched replicas showed that both the head and basal portions of the pillar cell were composed of a filamentous network consisting of several kinds of fibrous elements, into which numerous microtubules and actin filaments were tightly inserted. Myosin S1-decoration showed that the main constituent element in such filamentous networks in the basal portion of the pillar cell was the actin and tiny cross-bridges interconnected the randomly oriented adjacent actin filaments.     

Preservation and visualization of actin-containing filaments in the apical zone of cochlear sensory cells

The fine filamentous structure in the apical zone of cochlear sensory cells of the guinea pig was investigated under transmission electron microscopy (TEM) using various fixation methods. The true form of this structure, which is that of a dense core of sensory hairs and cuticular plates containing hair rootlets, has been hitherto unknown because of the selectively destructive effect of ordinary fixatives. We revealed the fine filamentous structure in great detail by fixing the specimens in tannic acid or by the modified glutaraldehyde-osmium fixation method, which can preserve actin filaments during the procedures required to prepare the specimen for TEM. The filamentous structure gives the impression of a negatively stained image when prepared in this way. Filaments were packed regularly and tightly into dense cores which projected down deep into the cuticular plate as hair rootlets. Cross-striations were seen at intervals of 360 ± 28 Å along the packed filaments, a distance which is comparable to the periodicity of an actin paracrystal. The overall diameter of each filament was 83 Å. In fact, the structure of dense cores and hair rootlets proved to be composed of actin paracrystals, probably containing some regulatory proteins. Cross-sectioned actin filaments in the paracrystal were arranged in an extremely regular hexagonal pattern. The characteristic filamentous texture in the cuticular plate was best seen in tissues that were pretreated with EDTA, and then fixed by tannic acid. It is probable that the greater part of the cuticular plate is composed of actin filaments and actin monomers, both containing Ca²⁺-dependent regulatory proteins. Utilizing the above ultrastructural findings, some functional models of this zone are proposed.     

Microtubules of guinea pig cochlear epithelial cells

By tannic acid staining, the 13-protofilament composition of cochlear hair cell microtubules, an impressive contrast against the 15-protofilament microtubules in cochlear pillar cells, was verified. The 15-protofilament microtubules formed a large and stiff cytoskeletal bundle in pillar cell bodies involving abundant actin filaments. The bundles were always situated vertically, i.e., longitudinally to the cell body axis, and were most numerous in the outer as well as the inner pillar cells in the basal turn, decreasing gradually toward the apex. Such gradient architecture of the pillar cell cytoskeleton can be correlated with the tuning mechanism for traveling waves of sound containing variable frequencies.     

Three-dimensional visualizations of the inner ear hair cell of the guinea pig. A rapid-freeze, deep-etch study of filamentous and membranous organelles

The fine structure of the filamentous and membranous organelles in the stereocilia and in the cuticular plate of sensory hair cells from the guinea pig was examined using a rapid-freeze, deep-etch method. In fixed and unfixed tissue the outer surface of the plasma membrane of the stereocilia had numerous surface protrusions of various sizes and shapes, while the protoplasmic fractured face of the membrane had rather sparse intramembrane particles. Many tiny cross links were present between the adjacent actin filaments and between actin filaments and the plasma membrane of the stereocilia. Numerous fibrils radiating from the hair rootlet were attached to the peripheral actin filaments in the cuticular plate. The radiating fibrils differed from the tiny cross links which interconnected the adjacent, randomly-oriented actin filaments in the cuticular plate. These complex structures consisting of actin filaments in the hair rootlets, radiating fibrils, and peripheral actin filaments may play an important role in regulating stereociliary bending.     

Development of hair cell stereocilia in the avian cochlea

The establishment of an embryonic hair cell's stereociliary bundle involves the coordinated regulation of several morphogenetic events. The developing hair cell organizes the assembly of individual stereocilia, regulates the growth of the stereociliary bundle, and aligns the orientation of the bundle. During development, individual stereocilia exhibit three phases of growth: (1) an initial assembly and elongation of a small number of actin filaments; (2) the development of the stereocilia rootlet and the addition of more filaments to each stereocilium; (3) a second growth phase where elongation of the actin filaments resumes. These three phases involve different biochemical conditions for actin assembly and, thus, are temporally separated during development. Each hair cell also regulates the size of its stereociliary bundle so that it fits into the precise basal-to-apical gradient in stereocilia length and width seen in the mature cochlea. Orientation of the stereociliary bundles also changes during development. Very young hair cells exhibit a non-uniform orientation. Early in development, neighboring groups of cells rapidly acquire a uniform orientation. A more gradual shift in orientation continues throughout development, so that by maturity most of the hair cells are oriented toward the abneural edge of the sensory epithelium.     

Immunofluorescence localization of proteins in semithin 0.2–1 μm frozen sections of the ear

Summary The present work describes a high resolution technique for locating proteins in frozen sections of the inner ear by immunofluorescence. Dissected organs are encapsulated in gelatin, and sections 0.1–1 m thick are cut at –100°C in a cryoultramicrotome. These are labelled with antibodies against two cytoskeletal proteins, actin and tubulin. Actin, which had previously only been described in the sensory cells, is found in the supporting cells as well. Tubulin is identified in the supporting cells and in outer spiral nerve fibres.Supported by grants from the Swedish Medical Research Council (no. 04x-02461), the Ragnar and Torsten Söderberg Foundation, and the Foundation Tysta Skolan     

Sensory hairs and filaments rods in vestibular hair cells of the waltzing guinea pig. Organization and identification of actin

The waltzing guinea pig suffers from hereditary deafness and vestibular disorder. In vestibular organs, hair cells of Type I develop pathologically and will eventually degenerate. They show fusion of sensory hairs, protrusion of the cuticular plate and contain a rod-shaped inclusion body. With fixation techniques designed to preserve proteins it is shown that this rod has a filamentous substructure reminding one of stereocilia. The packing density of the filaments is similar and circular packing patterns are seen within both structures. However, the rod has an irregular cross-section, as opposed to the circular circumference of stereocilia. The filaments in the rod were identified as containing the protein actin (as those in the stereocilia) by decoration with sub-fragment S-1 of myosin. All filaments in the rod have an identical functional polarity, pointing up from the nucleus towards the cuticular plate. This is contrary to that seen in stereocilia, which have filaments pointing down towards the cuticular plate. It is concluded that the rod is not developed by random polymerization of actin but is the result of co-ordinated assembly reminiscent of that which gives rise to stereocilia. The genetic defect appears to be related to mechanisms which determine the site of nucleation and the functional orientation of actin filaments during development.     

Synthesis of glycocalyx and associated structures in vestibular sensory cells

The ultrastructure of the glycocalyx with special reference to the synthesizing process was studied in the guinea pig vestibular sensory cells using the tannic acid staining technique. The glycocalyx emerged from the outer layer of the plasma membrane covering the entire length of the cilia. This glycocalyx also interconnected the ciliary structures tightly, such that a structural continuity was established between actin-membrane links and the glycocalyx. Interconnections between the actin filaments themselves were also noticed in the stereocilia as well as interconnections between individual actin filaments and the plasma membrane. These findings indicate that the glycocalyx and the ciliary interconnections may be closely related to the sensory hair transduction system. In the cellular cytoplasm, vesicles seemingly related to the synthesis of the glycocalyx were observed. These coated vesicles, which were synthesized by the Golgi complex and endoplasmic reticulum, interacted with the plasma membrane forming a coated pit. The lysosomal-like bodies also observed in the cell were closely related to the glycocalyx as well. Thus the glycocalyx seems to be synthesized by the endoplasmic reticulum and Golgi complexes and transferred through the coated vesicles or lysosomal-like bodies to the apical plasma membrane.     

Structural basis for mechanical transduction in the frog vestibular sensory apparatus: I. The otolithic membrane

The mechanical coupling of the otoliths to the hair cell sensory stereocilia at the surface of the vestibular sensory epithelium is mediated by two layers of extracellular matrix, each one with a specific role in the mechanical transduction process. The first is a rigid layer in direct contact with the otolithic mass and is known as the otolithic membrane or gelatin membrane. This structure consists of a dense, randomly cross linked filament network that uniformly distributes the force of inertia of the non-uniform otolithic mass to all stereocilia bundles. The second layer formed by a columnar organization of filaments secures the otolithic membrane above the surface of the epithelium. The long columnar filaments are organized in parallel to the stereocilia bundles and are anchored to the apical surface of the supporting cells. The zonula adherens at the apical region of each supporting cell displays a thick polygonal bundle of actin filaments forming at the surface of the epithelium a transcellular honeycomb organization that provides mechanical ground support for the columnar filament layer. The dominant aspect of this columnar filament layer indicates that it may also have an important role in attenuating the force of inertia of the large otolithic mass during acceleration, screening stresses that would be directed to an effective bending of the stereocilia bundles.     

Morphometric analysis of the vestibular sensory epithelia of young adult rat

The appearance of vestibular sensory cells and their progressive development has been the subject of many ontogenetic studies. Because deteriorating hair cells are supposed to play a role in balance disorders of the elderly, the final stage of development (i.e. senescence) has been investigated as well. It is generally assumed that the number of hair cells in crista ampullaris, saccule and utricle slowly but steadily decreases with age. However, actual data covering the period between maturation and senescence are scarce. In the present study, rat vestibular epithelia were labeled for actin and tubulin. Morphology was inspected from immediately after weaning until the age of 12 months. Although, postnatal development was no part of this study some data on one day old epithelia are presented for comparison. At postnatal day 1, hair bundles are still shorter than in mature sensory organs, the width of the zonula adherens is less, and the apical cross-sectional area of the epithelial cells is smaller. After one month, maturation is complete. Total cell density is 400-500 per 0.01 mm2, both in the otolith maculae and in the cristae ampullares. During the first year after maturation, no changes in epithelial morphology were observed and cell density remains constant.     

Selective Expression of β Tubulin Isotypes in Gerbil Vestibular Sensory Epithelia and Neurons

The seven mammalian isotypes of tubulin are strikingly similar in amino acid sequence. The differences in isotypic sequence, although small, are nonetheless conserved in evolution, which suggests that they may confer distinct functional roles. If so, such roles should be reflected in the selective expression of isotypes by cell type, or even in the sorting of isotypes to within-cell pools. Hair cells of the vestibular sensory epithelia each possess a kinocilium, a microtubule-based organelle that could represent a distinct microtubule compartment, separate from the extensive microtubule network in the soma. The afferent neurons that innervate the vestibular sensory epithelia may also be functionally divided into dendritic, somatic, and axonal compartments, each with its own complement of microtubules. We have examined the distribution of tubulin isotypes in gerbil vestibular epithelia using isotype-specific antibodies to four isotypes and indirect immunofluorescence. We found that hair cells selectively express _I and _IV tubulin, while supporting cells express _I, _II, and _IV tubulin. However, no sorting of isotypes between somatic and kinocilia compartments was found in hair cells. Vestibular ganglion cells display three isotypes in the soma, axon, and terminal dendrite compartments (_I, _II, and _III tubulin), but only _III tubulin was found in calyceal nerve endings. The implication of these findings is that tubulin isotypes are not sorted to within-cell compartments in hair cells but are sorted in some vestibular neurons.     

Closure of supporting cell scar formations requires dynamic actin mechanisms

In many vertebrate inner ear sensory epithelia, dying sensory hair cells are extruded, and the apices of surrounding supporting cells converge to re-seal the epithelial barrier between the electrochemically-distinct endolymph and perilymph. These cellular mechanisms remain poorly understood. Dynamic microtubular mechanisms have been proposed for hair cell extrusion; while contractile actomyosin-based mechanisms are required for cellular extrusion and closure in epithelial monolayers. The hypothesis that cytoskeletal mechanisms are required for hair cell extrusion and supporting cell scar formation was tested using bullfrog saccules incubated with gentamicin (6h), and allowed to recover (18h). Explants were then fixed, labeled for actin and cytokeratins, and viewed with confocal microscopy. To block dynamic cytoskeletal processes, disruption agents for microtubules (colchicine, paclitaxel) myosin (Y-27632, ML-9) or actin (cytochalasin D, latrunculin A) were added during treatment and recovery. Microtubule disruption agents had no effect on hair cell extrusion or supporting cell scar formation. Myosin disruption agents appeared to slow down scar formation but not hair cell extrusion. Actin disruption agents blocked scar formation, and largely prevented hair cell extrusion. These data suggest that actin-based cytoskeletal processes are required for hair cell extrusion and supporting cell scar formation in bullfrog saccules.     

Tubulin expression in the developing and adult gerbil organ of Corti

In the late stages of inner ear development, the relatively undifferentiated cells of Kollicker's organ are transformed into the elaborately specialized cell types of the organ of Corti. Microtubules are prominent features of adult cells in the organ of Corti, particularly supporting cells. To test the possible role of microtubules in organ of Corti development, the microtubule organization in the organ of Corti has been examined using indirect immunofluorescence to beta-tubulin in the developing gerbil cochlea. Tubulin first appears at post-natal day 0 (P0) as filamentous asters in inner hair cells and by P2, asters are also seen in outer hair cells. Tubulin appears at P3 in inner pillar cells in a tooth crown-like figure. By P6, tubulin expression is also evident in outer pillar cells and by P9, it is seen in Deiters cells. Elaboration of microtubules in pillar cells was observed to proceed from the reticular lamina towards the basilar membrane. The pattern of tubulin expression in the apical organ of Corti lags the base by about 3 days until P6, but by P9, apical and basal organ of Corti appear substantially the same.     

Tubulin and microtubules in cochlear hair cells: comparative immunocytochemistry and ultrastructure

The distribution of tubulin has been investigated in surface preparations of the guinea pig organ of Corti using indirect immunofluorescence microscopy. Two different monoclonal antibodies to tubulin produce similar distinct patterns of labelling in hair cells. Labelling is greater in inner hair cells than outer hair cells. It occurs in rings around the cell apex, and in a meshwork below and channels through, the cuticular plate. In outer hair cells from the apical region of the cochlea, labelling occurs around the location of a basalward protrusion of the cuticular plate. These patterns correlate with the location of microtubules observed using transmission electron microscopy. A large patch of labelling occurs on the strial side of the cell corresponding to the largest channel through the cuticular plate and the kinociliary basal body. Strands of labelling are seen running parallel to the long axis of the cell between the subcuticular and synaptic region. Many more of these strands are seen in the inner hair cell than the outer hair cell and may correspond to tracks of microtubules transporting neurotransmitter vesicles or other organelles. In outer hair cells, intense labelling and many microtubules are seen in the subnuclear region. The possible roles of the different microtubule arrangements are discussed.