Actin filaments, stereocilia, and hair cells of the bird cochlea. II. Packing of actin filaments in the stereocilia and in the cuticular plate and what happens to the organization when the stereocilia are bent

A comparison of hair cells from different parts of the cochlea reveals the same organization of actin filaments; the elements that vary are the length and number of the filaments. Thin sections of stereocilia reveal that the actin filaments are hexagonally packed and from diffraction patterns of these sections we found that the actin filaments are aligned such that the crossover points of adjacent actin filaments are in register. As a result, the cross-bridges that connect adjacent actin filaments are easily seen in longitudinal sections. The cross-bridges appear as regularly spaced bands that are perpendicular to the axis of the stereocilium. Particularly interesting is that, unlike what one might predict, when a stereocilium is bent or displaced, as might occur during stimulation by sound, the actin filaments are not compressed or stretched but slide past one another so that the bridges become tilted relative to the long axis of the actin filament bundle. In the images of bent bundles, the bands of cross- bridges are then tilted off perpendicular to the stereocilium axis. When the stereocilium is bent at its base, all cross-bridges in the stereocilium are affected. Thus, resistance to bending or displacement must be property of the number of bridges present, which in turn is a function of the number of actin filaments present and their respective lengths. Since hair cells in different parts of the cochlea have stereocilia of different, yet predictable lengths and widths, this means that the force needed to displace the stereocilia of hair cells located at different regions of the cochlea will not be the same. This suggests that fine tuning of the hair cells must be a built-in property of the stereocilia. Perhaps its physiological vulnerability may result from changes of stereociliary structure.     

Actin filaments, stereocilia, and hair cells of the bird cochlea. III. The development and differentiation of hair cells and stereocilia

The cochleae of chick embryos of 8 days of incubation until hatching (21 days) were examined by scanning electron microscopy. Unlike what one would expect from the literature, the total number of hair cells per cochlea (10,405 +/- 529) is already determined and visible in a 10-day embryo and the growth of the cochlea is a result of the growth in size and surface area of the hair cells. We also find that the hair cells differentiate simultaneously throughout the cochlea and have followed the differentiation of individual hair cells throughout development. During development we find that the total number, hexagonal packing, and orientation of the stereocilia in each hair cell is determined early and accurately (9- to 10-day embryos). The stereocilia then begin to elongate in all the cells of the cochlea at approximately 0.5 micron/day. By Day 12 the tallest stereocilia in each cell are 1.5-1.8 micron long, the mature length for cells at the proximal end of the cochlea. At this point all stereocilia cease elongating, but those along the inferior edge gradually increase in width from 0.11 micron to maximally 0.19 micron in 17-day embryos. When the stereocilia on the inferior edge reach their mature width, widening ceases and the elongation of stereocilia in the distal hair cells begins again. When these stereocilia have attained their mature lengths, they stop growing. Thus elongation and widening of stereocilia are separated in time. During this period, 11 to 13 days, the shape of the tufts at the proximal end of the cochlea changes. This occurs because stereocilia in the front of each tuft are absorbed while others at the sides appear de novo. This rearrangement converts a circular bundle of stereocilia to a rectangular bundle.     

Actin filaments, stereocilia and hair cells of the bird cochlea. VI. How the number and arrangement of stereocilia are determined.

Beginning in 8-day embryos, stereocilia sprout from the apical surface of hair cells apparently at random. As the embryo continues to develop, the number of stereocilia increases. By 10 1/2 days the number is approximately the same as that encountered extending from mature hair cells at the same relative positions in the adult cochlea. Surprisingly, over the next 2-3 days the number of stereocilia continues to increase so that hair cells in a 12-day embryo have 1 1/2 to 2 times as many stereocilia as in adult hair cells. In short, there is an overshoot in stereociliary number. During the same period in which stereocilia are formed (9-12 days) the apical surface of each hair cell is filled with closely packed stereocilia; thus the surface area is proportional to the number of stereocilia present per hair cell, as if these features were coupled. The staircase begins to form in a 10-day embryo, with what will be the tallest row beginning to elongate first and gradually row after row begins to elongate by incorporation of stereocilia at the foot of the staircase. Extracellular connections or tip linkages appear as the stereocilia become incorporated into the staircase. After a diminutive staircase has formed, eg. in a 12-day embryo, the remaining stereocilia located at the foot of the staircase begin to be reabsorbed, a process that occurs during the next few days. We conclude that the hair cell determines the number of stereocilia to form by filling up the available apical surface area with stereocilia and then, by cropping back those that are not stabilized by extracellular linkages, arrives at the appropriate number. Furthermore, the stereociliary pattern, which changes from having a round cross-sectional profile to a rectangular one, is generated by these same linkages which lock the stereocilia into a precise pattern. As this pattern is established, we envision that the stereocilia flow over the apical surface until frozen in place by the formation of the cuticular plate in the apical cell cytoplasm.     

Actin filaments, stereocilia, and hair cells of the bird cochlea. V. How the staircase pattern of stereociliary lengths is generated

The stereocilia on each hair cell are arranged into rows of ascending height, resulting in what we refer to as a "staircase-like" profile. At the proximal end of the cochlea the length of the tallest row of stereocilia in the staircase is 1.5 micron, with the shortest row only 0.3 micron. As one proceeds towards the distal end of the cochlea the length of the stereocilia progressively increases so that at the extreme distal end the length of the tallest row of the staircase is 5.5 micron and the shortest row is 2 micron. During development hair cells form their staircases in four phases of growth separated from each other by developmental time. First, stereocilia sprout from the apical surfaces of the hair cells (8-10-d embryos). Second (10-12-d embryos), what will be the longest row of the staircase begins to elongate. As the embryo gets older successive rows of stereocilia initiate elongation. Thus the staircase is set up by the sequential initiation of elongation of stereociliary rows located at increased distances from the row that began elongation. Third (12-17-d embryos), all the stereocilia in the newly formed staircase elongate until those located on the first step of the staircase have reached the prescribed length. In the final phase (17-d embryos to hatchlings) there is a progressive cessation of elongation beginning with the shortest step and followed by taller and taller rows with the tallest step stopping last. Thus, to obtain a pattern of stereocilia in rows of increasing height what transpires are progressive go signals followed by a period when all the stereocilia grow and ending with progressive stop signals. We discuss how such a sequence could be controlled.     

Actin filaments, stereocilia, and hair cells of the bird cochlea. I. Length, number, width, and distribution of stereocilia of each hair cell are related to the position of the hair cell on the cochlea

Located on the sensory epithelium of the sickle-shaped cochlea of a 7- to 10-d-old chick are approximately 5,000 hair cells. When the apical surface of these cell is examined by scanning microscopy, we find that the length, number, width, and distribution of the stereocilia on each hair cell are predetermined. Thus, a hair cell located at the distal end of the cochlea has 50 stereocilia, the longest of which are 5.5 microns in length and 0.12 microns in width, while those at the proximal end number 300 and are maximally 1.5 microns in length and 0.2 micron in width. In fact, if we travel along the cochlea from its distal to proximal end, we see that the stereocilia on successive hair cells gradually increase in number and width, yet decrease in length. Also, if we look transversely across the cochlea where adjacent hair cells have the same length and number of stereocilia (they are the same distance from the distal end of the cochlea), we find that the stereocilia of successive hair cells become thinner and that the apical surface area of the hair cell proper, not including the stereocilia, decreases from a maximum of 80 microns2 to 15 microns2. Thus, if we are told the length of the longest stereocilium on a hair cell and the width of that stereocilium, we can pinpoint the position of that hair cell on the cochlea in two axes. Likewise, if we are told the number of stereocilia and the apical surface of a hair cell, we can pinpoint the location of that cell in two axes. The distribution of the stereocilia on the apical surface of the cell is also precisely determined. More specifically, the stereocilia are hexagonally packed and this hexagonal lattice is precisely positioned relative to the kinocilium. Because of the precision with which individual hair cells regulate the length, width, number, and distribution of their cell extensions, we have a magnificent object with which to ask questions about how actin filaments that are present within the cell are regulated. Equally interesting is that the gradient in stereociliary length, number, width, and distribution may play an important role in frequency discrimination in the cochlea. This conclusion is amplified by the information presented in the accompanying paper (Tilney, L.G., E.H. Egelman, D.J. DeRosier, and J.C. Saunders, 1983, J. Cell Biol., 96:822- 834) on the packing of actin filaments in this stereocilia.     

Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochlea

The sensory epithelium of the chick cochlea contains only two cell types, hair cells and supporting cells. We developed methods to rapidly dissect out the sensory epithelium and to prepare a detergent-extracted cytoskeleton. High salt treatment of the cytoskeleton leaves a "hair border", containing actin filament bundles of the stereocilia still attached to the cuticular plate. On SDS-PAGE stained with silver the intact epithelium is seen to contain a large number of bands, the most prominent of which are calbindin and actin. Detergent extraction solubilizes most of the proteins including calbindin. On immunoblots antibodies prepared against fimbrin from chicken intestinal epithelial cells cross react with the 57- and 65-kD bands present in the sensory epithelium and the cytoskeleton. It is probable that the 57-kD is a proteolytic fragment of the 65-kD protein. Preparations of stereocilia attached to the overlying tectorial membrane contain the 57- and 65-kD bands. A 400-kD band is present in the cuticular plate. By immunofluorescence, fimbrin is detected in stereocilia but not in the hair borders after salt extraction. The prominent 125 A transverse stripping pattern characteristic of the actin cross-bridges in a bundle is also absent in hair borders suggesting fimbrin as the component that gives rise to the transverse stripes. Because the actin filaments in the stereocilia of hair borders still remain as compact bundles, albeit very disordered, there must be an additional uncharacterized protein besides fimbrin that cross-links the actin filaments together.     

The organization of actin filaments in the stereocilia of cochlear hair cells

Within each tapering stereocilium of the cochlea of the alligator lizard is a bundle of actin filaments with > 3,000 filaments near the tip and only 18-29 filaments at the base where the bundle enters into the cuticular plate; there the filaments splay out as if on the surface of a cone, forming the rootlet. Decoration of the hair cells with subfragment 1 of myosin reveals that all the filaments in the stereocilia, including those that extend into the cuticular plate forming the rootlet, have unidirectional polarity, with the arrowheads pointing towards the cell center. The rest of the cuticular plate is composed of actin filaments that show random polarity, and numerous fine, 30 A filaments that connect the rootlet filaments to each other, to the cuticular plate, and to the membrane. A careful examination of the packing of the actin filaments in the stereocilia by thin sectin and by optical diffraction reveals that the filaments are packed in a paracrystalline array with the crossover points of all the actin helices in hear-perfect register. In transverse sections, the actin filaments are not hexagonally packed but, rather, are arranged in scalloped rows that present a festooned profile. We demonstrated that this profile is a product of the crossbridges by examining serial sections, sections of different thicknesses, and the same stereocilium at two different cutting angles. The filament packing is not altered by fixation in different media, removal of the limiting membrane by detergent extraction, or incubation of extracted hair cells in EGTA, EDTA, and Ca++ and ATP. From our results, we conclude that the stereocilia of the ear, unlike the brush border of intestinal epithelial cells, are not designed to shorten, nor do the filaments appear to slide past one another. In fact, the stereocilium is like a large, rigid structure designed to move as a lever.     

The actin filament content of hair cells of the bird cochlea is nearly constant even though the length, width, and number of stereocilia vary depending on the hair cell location

By direct counts off scanning electron micrographs, we determined the number of stereocilia per hair cell of the chicken cochlea as a function of the position of the hair cell on the cochlea. Micrographs of thin cross sections of stereociliary bundles located at known positions on the cochlea were enlarged and the total number of actin filaments per stereocilium was counted and recorded. By comparing the counts of filament number with measurements of actin filament bundle width of the same stereocilium, we were able to relate actin filament bundle width to filament number with an error margin (r2) of 16%. Combining this data with data already published or in the process of publication from our laboratory on the length and width of stereocilia, we were able to calculate the total length of actin filaments present in stereociliary bundles of hair cells located at a variety of positions on the cochlea. We found that stereociliary bundles of hair cells contain 80,000-98,000 micron of actin filament, i.e., the concentration of actin is constant in all hair cells with a range of values that is less than our error in measurement and/or biological variation, the greatest variation being in relating the diameters of the stereocilia to filament number. We also calculated the membrane surface needed to cover the stereocilia of hair cells located throughout the cochlea. The values (172-192 micron 2) are also constant. The implications of our observation that the total amount of actin is constant even though the length, width, and number of stereocilia per hair cell vary are discussed.     

Actin, alpha-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells

During the spreading of a population of rat embryo cells, approximately 40% of the cells develop a strikingly regular network which precedes the formation of the straight actin filament bundles seen in the fully spread out cells. Immunofluorescence studies with antibodies specific for the skeletal muscle structural proteins actin, alpha-actinin, and tropomyosin indicate that this network is composed of foci containing actin and alpha-actinin, connected by tropomyosin-associated actin filaments. Actin filaments, having both tropomyosin and alpha-actinin associated with them, are also seen to extend from the vertices of this network to the edges of the cell. These results demonstrate a specific interaction of alpha-actinin and tropomyosin with actin filaments during the assembly and organization of the actin filament bundles of tissue culture cells. The three-dimensional network they form may be regarded as the structural precursor and the vertices of this network as the organization centers of the ultimately formed actin filament bundles of the fully spread out cells.     

Structure of outer hair cell stereocilia side and attachment links in the chinchilla cochlea.

The structure and symmetry of chinchilla outer hair cell (OHC) stereocilia side and attachment links were investigated by transmission electron microscopy using tannic acid and Cuprolinic blue histochemical procedures. The side links run laterally between and across the rows of the stereocilia and connect the stereocilia together within the bundle. Attachment links form a crown-like array around the tips of only the tallest OHC stereocilia and attach these stereocilia to the Type B fibrils of the tectorial membrane. Computer averaging of the side links from tannic acid-treated tissues showed a central dense region of the link between adjacent stereocilia and a smaller dense portion at the plasma membrane end of the link. Computer averaging of Cuprolinic blue-treated tissues showed low electron density of the central region of the link, and the plasma membrane ends of the link were electron dense. After tannic acid treatment, the attachment links showed a diffused radial distribution around the tips of the tallest OHC stereocilia. After Cuprolinic blue treatment, the attachment links appeared as electron-dense, membrane-bound granular structures arranged with radial symmetry. The central regions of the side links are reactive to tannic acid. These regions appear to contain neutral and basic residues of proteins and participate in side-by-side association of the side links in regular aggregates. Cuprolinic blue-reactive regions of the side and attachment links appear to contain acidic sulfated residues of glycoproteins or proteoglycans, which may be involved in the attachment of these links to the stereocilium membrane.     

Distribution and polarity of actin in the sensory hair cells of the chinchilla cochlea

Summary The distribution and polarity of actin in sensory hair cells of the chinchilla cochlea has been determined by decoration of actin filaments with myosin sub fragment S₁. Decorated actin filaments of the same polarity were present within the stereocilia above the cuticular plate. However the filaments in the rootlets and the thin filaments projecting laterally from the rootlets into the cuticular plate did not decorate with S₁. Decorated actin filaments were present within the cuticular plate, and near the plasma-membrane filaments of opposite polarity were observed. In the cross-striated region at the base of the cuticular plate of inner hair cells, decorated filaments were present in the dense bands of the cross-striations but the thin filaments perpendicular to the dense bands were not decorated. These results are discussed with respect to the two mechanisms that have been suggested for actin-myosin mediated movement of the stereocilia of inner-ear sensory cells.     

Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear

Replicas of the apical surface of hair cells of the inner ear (vestibular organ) were examined after quick freezing and rotary shadowing. With this technique we illustrate two previously undescribed ways in which the actin filaments in the stereocilia and in the cuticular plate are attached to the plasma membrane. First, in each stereocilium there are threadlike connectors running from the actin filament bundle to the limiting membrane. Second, many of the actin filaments in the cuticular plate are connected to the apical cell membrane by tiny branched connecting units like a "crow's foot." Where these "feet" contact the membrane there is a small swelling. These branched "feet" extend mainly from the ends of the actin filaments but some connect the lateral surfaces of the actin filaments as well. Actin filaments in the cuticular plate are also connected to each other by finer filaments, 3 nm in thickness and 74 +/- 14 nm in length. Interestingly, these 3-nm filaments (which measure 4 nm in replicas) connect actin filaments not only of the same polarity but of opposite polarities as documented by examining replicas of the cuticular plate which had been decorated with subfragment 1 (S1) of myosin. At the apicolateral margins of the cell we find two populations of actin filaments, one just beneath the tight junction as a network, the other at the level of the zonula adherens as a ring. The latter which is quite substantial is composed of actin filaments that run parallel to each other; adjacent filaments often show opposite polarities, as evidenced by S1 decoration. The filaments making up this ring are connected together by the 3-nm connectors. Because of the polarity of the filaments this ring may be a "contractile" ring; the implications of this is discussed.     

The structure of the cuticular plate, an in vivo actin gel

The cuticular plate is a network of actin filaments found in hair cells of the cochlea. In the alligator lizard, it consists of rootlets, emanating from the stereocilia, and of cross-connecting actin filaments that anchor these rootlets. In thin sections, this network displays striking patches of 650 +/- 110-A striae. By quantitative analyses of the images, the mystery of the striae can be explained. They are due in part to the rootlets which are sets of flat ribbons of actin filaments. The ribbons in each set are separated by approximately 650 A. Numerous whiskers 30 A in diameter extend from each ribbon's face, interconnecting adjacent ribbons. The nonrootlet filaments, except at the margins of the cell, occur primarily as single filaments. Like the ribbons, they are bristling with whiskers. The patches of striae are explained by ribbons and filaments held at a 650-A separation by the whiskers that project from them. A simple model for regions of bewhiskered filaments is a box crammed full of randomly oriented test- tube brushes. A thin slice through the box will show regions of dark lines or striae due to the wire backbones of the brushes separated from one another by the bristle length. Using the computer instead of test- tube brushes, we have been able to model quantitatively the filament distribution and pattern of striae seen in the cuticular plate of the lizard. The organization of actin filaments we have deduced from our simulations differs from that found in macrophages or in the terminal web of intestinal epithelial cells.     

Transient expression of the t-isoform of plastins/fimbrin in the stereocilia of developing auditory hair cells

The transduction of auditory signals by cochlear hair cells depends upon the integrity of hair cell stereociliary bundles. Stereocilia contain a central core of actin filaments, cross-linked by actin bundling proteins. In the cochlea, the two proteins described to date as responsible for the spatial arrangement of actin filaments in sterocilia are fimbrin and the recently discovered espin. Fimbrin (the chick homolog of human I-plastin) belongs to the plastins/fimbrin family that includes two additional isoforms of plastins, T- and L-plastin. In the present study, we used isoform specific antibodies to investigate the presence of the T- and L-isoforms of plastin/fimbrin in the adult and developing rat cochlea. We found that T-plastin, but not L-plastin, is expressed in the rat cochlea. During postnatal development of the rat organ of Corti, T-plastin can be detected in the core of stereocilia from early stages of hair cell differentiation, and its expression gradually increases in stereocilia as hair cells mature. However, as opposed to other actin-binding proteins expressed in stereocilia, T-plastin is absent from the stereocilia of mature hair cells. Such temporally restricted expression strengthens the idea of functional differences between plastins isoforms, and suggests that T-plastin could have a specific role in stereocilia formation.     

Microtubules regulate the organization of actin filaments at the cortical region in root hair cells ofHydrocharis

Summary We studied the mechanism controlling the organization of actin filaments (AFs) inHydrocharis root hair cells, in which reverse fountain streaming occurs. The distribution of AFs and microtubules (MTs) in root hair cells were analyzed by fluorescence microscopy and electron microscopy. AFs and MTs were found running in the longitudinal direction of the cell at the cortical region. AFs were observed in the transvacuolar strand, but not MTs. Ultrastructural studies revealed that AFs and MTs were colocalized and that MTs were closer to the plasma membrane than AFs. To examine if MTs regulate the organization of AFs, we carried out a double inhibitor experiment using cytochalasin B (CB) and propyzamide, which are inhibitors of AFs and MTs, respectively. CB reversibly inhibited cytoplasmic streaming while propyzamide alone had no effect on it. However, after treatment with both CB and propyzamide, removal of CB alone did not lead to recovery of cytoplasmic streaming. In these cells, AFs showed a meshwork structure. When propyzamide was also removed, cytoplasmic streaming and the original organization of AFs were recovered. These results strongly suggest that MTs are responsible for the organization of AFs inHydrocharis root hair cells.     

Tetrodotoxin-sensitive, voltage-dependent sodium currents in hair cells from the alligator cochlea.

We have used whole-cell patch clamp techniques to record from tall hair cells isolated from the apical half of the alligator cochlea. Some of these cells gave action potentials in response to depolarizing current injections. When the same cells were voltage clamped, large transient inward currents followed by smaller outward currents were seen in response to depolarizing steps. We studied the transient inward current after the outward current had been blocked by external tetraethylammonium (20 mM) or by replacing internal potassium with cesium. It was found to be a sodium current because it was abolished by either replacing external sodium with choline or by external application of tetrodotoxin (100 nM). The sodium current showed voltage-dependent activation and inactivation. Most of the spiking hair cells came from the apex of the cochlea, where they would be subject to low-frequency mechanical stimulation in vivo.     

Roles of actin filaments in cytoplasmic streaming and organization of transvacuolar strands in root hair cells ofHydrocharis

Summary Effects of cytochalasin B and mycalolide-B on cytoplasmic streaming, organizations of actin filaments and the transvacuolar strand were studied in root hair cells ofHydrocharis, which shows reverse fountain streaming. Both toxins inhibited cytoplasmic streaming and destroyed the organizations of actin filaments and transvacuolar strands. However, we found a great difference between these toxins with respect to reversibility. The effects of cytochalasin B were reversible but not those of mycalolide B. The present results suggest that actin filaments work as a track of cytoplasmic streaming and as a cytoskeleton to maintain the transvacuolar strand. The usefulness of root hair cells ofHydrocharis in studying the dynamic organization of actin filaments of plant is discussed.Abbreviations CB cytochalasin B - DMSO dimethylsulfoxide - ML-B mycalolide B