The dimensions and composition of stereociliary rootlets in mammalian cochlear hair cells: comparison between high- and low-frequency cells and evidence for a connection to the lateral membrane

Abstract

The sensory bundle of vertebrate cochlear hair cells consists of actin-containing stereocilia that are thought to bend at their ankle during mechanical stimulation. Stereocilia have dense rootlets that extend through the ankle region to anchor them into the cuticular plate. Because this region may be important in bundle stiffness and durability during prolonged stimulation at high frequencies, we investigated the structure and dimensions of rootlets relative to the stereocilia in apical (low-frequency) and basal (high-frequency) regions of rodent cochleae using light and electron microscopy. Their composition was investigated using postembedding immunogold labeling of tropomyosin, spectrin, beta-actin, gamma-actin, espin, and prestin. The rootlets have a thick central core that widens at the ankle, and are embedded in a filamentous meshwork in the cuticular plate. Within a particular frequency region, rootlet length correlates with stereociliary height but between regions it changes disproportionately; apical stereocilia are, thus, approximately twice the height of basal stereocilia in equivalent rows, but rootlet lengths increase much less. Some rootlets contact the tight junctions that underlie the ends of the bundle. Rootlets contain spectrin, tropomyosin, and beta- and gamma-actin, but espin was not detected; spectrin is also evident near the apical and junctional membranes, whereas prestin is confined to the basolateral membrane below the junctions. These data suggest that rootlets strengthen the ankle region to provide durability and may contact with the lateral wall either to give additional anchoring of the stereocilia or to provide a route for interactions between the bundle and the lateral wall.

Figure 1.

A, Scanning electron micrograph of an isolated guinea pig OHC showing the lip in the apical region (arrows). B, Detail of the lip (arrows) of the OHC shown in A. Inset, Single-plane confocal image showing the apical lip of an unfixed (living) guinea pig OHC after visualization with calcium green. C, A guinea pig basal OHC showing that the lip (arrow) is less pronounced. Arrowheads show where the stereocilia at the end of the W overhang the cell body because of the lip. D, Horizontal transmission electron microscopy section of a rat OHC showing the W-shape formed by the rootlets (in the center) and stereociliary ankles (at the end of one arm). Tall (t), intermediate (i), and short (s) rows can be identified with additional rootlet-like profiles inside the W where no matching stereocilia were found in serial sections (arrow). The arrowheads point to a line of three rootlets where it is possible to see them getting thinner from tall to short rows. The W is not central, but lies closer to the modiolar side so that the ends of the arms are likely to overlie the lip. E, Outline of a horizontal section from the basal half of the cochlea through the apex and bundle (red) of a guinea pig OHC superimposed on the lower cell body showing how the stereocilia extend beyond the subcuticular boundaries of the cell. Scale bars: A, 10 μm; B, C, 2.5 μm; D, 1 μm; E, 2 μm.

Figure 2.

Structure of the rootlets in rat OHCs. A, Typical example of a hair bundle in a 40 nm vertical ultrathin section from an apical location. The tall (t), intermediate (i), and short (s) stereocilia are visible and rootlets (r) of the tall and intermediate rows; the short rootlet is out of the plane of section. A lighter zone is apparent around the rootlet (asterisk) and splaying is visible at the lower end of the intermediate rootlet (arrow). B, Similar view in a semithin (250 nm) section which has captured the full width and length of the rootlets (as determined from adjacent serial sections). Note the tapering of the rootlet into the shaft, particularly of the tall stereocilium (arrowhead), and the splaying of the end of the tallest stereociliary rootlet in the cuticular plate (arrow). C, The ankle region of a tall stereocilium, showing the filaments in the stereocilium shaft converging onto the rootlet (arrow shows this on one side). Inset, A cross section of a rootlet deep in the cuticular plate showing the crescent shaped profile. D, Serial sections of a single stereocilium through the ankle region. The rootlet is the dense central material. E, Example of a rootlet extending through the lower edge of the cuticular plate (arrow) into the apical cytoplasm, where it splays out. Scale bars: A–C, inset, 200 nm; D, 100 nm; E, 150 nm.

Figure 3.

A, Top, Cross section of a guinea pig tall stereocilium showing the central core of the rootlet and a peripheral dense ring around it (arrowheads). Bottom two panels show two sections of a rat OHC stereocilium from a series used to create the 3D reconstruction in B. The top of the two is at the level of the ankle region, which is virtually filled with dense material. The bottom of the two is at the level of the cuticular plate, showing that the rootlet has narrowed. Scale bar, 100 nm. B, 3D reconstruction of the rootlet in the ankle region. The central core is joined by peripheral dense filaments of material (e.g., arrowheads) that may coat converging actin filaments. These sheaths around the filaments end above the level of the apical membrane. Scale bar, 100 nm. C, Cross sectional area in nanometers squared of the rootlet material in five different sets of cross sections spanning ∼900 nm through the ankle region of two tall, two intermediate, and one short stereocilium from a rat OHC. Each section is represented by a single black bar except for the section representing the upper surface of the cuticular plate, shown in red. The thickest part of the rootlet is usually just at the entry into the apical membrane and the cross sectional area decreases across the tall to short rows.

Figure 4.

Serial section reconstruction of representative stereocilia from an apical hair bundle. A, Scanning electron micrograph of a guinea pig OHC bundle to illustrate the concept of a “column” of stereocilia (colored red). B, Two sequential sections of the nine semithin (200 nm) serial sections of a rat OHC used to construct a column of stereocilia as illustrated in A. Note the dense rootlet like material in the upper region of the tall stereocilium displaced toward the edge (arrow). This is discontinuous with the normal rootlet material. C, Stereopair of the reconstructed column. Note how the thickness and length of the rootlet is approximately proportional to the height of the stereocilia in each row. The upper dense material has been omitted for clarity. Scale bars, 200 nm.

Figure 5.

Histogram showing the mean lengths of stereocilia and cuticular portion of the rootlets from apical and basal hair cells from rat cochlea. Means and SEMs are given for each row of stereocilia from each row of OHCs and are based on all of the stereocilia and rootlets measured for the present study in three animals. Comparison of apical and basal hair cells shows that although the stereocilia are substantially shorter in the basal regions compared with their apical counterparts, the rootlets change to a much lesser degree. Data for IHCs derived from one animal are shown for comparison and reveal a similar pattern. t, Tall; i, intermediate; s, short; gray upward bars are the stereocilia; white downward bars are the rootlets.

Figure 6.

Scatterplot of all of the complete stereocilia–rootlet pairs measured in this study from the rat cochleae. Apical data from two of the three animals (denoted by diamonds or triangles) are plotted with black symbols and blue and green trend lines respectively; basal data from the same two animals (denoted again by diamonds, triangles) are plotted as red symbols with pink and purple trend lines respectively. Additional data from a third animal for the basal region are plotted in red squares with a red trend line. In both apical and basal data, the height of stereocilia and length of rootlets are correlated, as indicated by the trend lines. However, the slopes of the trend lines for the basal data are shallower than for the apical data. The three different rows of stereocilia–rootlet pairs are not easily distinguishable in basal data when measurements from all animals are plotted on the same graph, because they partially overlap, but can be clearly seen in the apical data where they are circled and labeled. t, Tall; i, intermediate; s, short.

Figure 7.

A, Vertical ultrathin section of an apical rat OHC showing a row of rootlets sectioned obliquely, one of which joins the lower end of the lateral junctional complex (arrow). Note the lip around the cell apex (arrowheads). B, Semithin section of a basal rat OHC showing contact between the rootlet of the tall stereocilia (t) and the lateral wall (arrow). Intermediate stereocilia (i) and short stereocilia (s) rootlets also closely approach the wall. C, Another basal rat OHC where short stereociliary rootlets contact the lateral wall (arrow). Intermediate stereocilia are also visible (i). D, Horizontal section of an apical rat hair cell showing the rootlets on either end of the bundle closely in contact with the lateral wall (arrows). E, Detail of a serial section to that shown in D. Rootlets approach the junctional complex and some appear to have filaments extending into it (arrow). F, Vertical section of a rootlet contacting the lateral wall in a rat hair cell. The dense material appears to be contiguous. Scale bars: A, 1 μm; B–D, 0.5 μm; E, F, 100 nm.

Figure 8.

The structure of the meshwork in the cuticular plate. A, Horizontal section of a guinea pig OHC. The dense material forms a mesh around the rootlets extending over the superficial aspect of the plate. Channels in the periphery of the cuticular plate (arrows) and at the apex of the W (c) are visible. B, Vertical section of a rat OHC, showing vertically orientated parallel actin filaments between the rootlets (arrow). C, A subapical dense layer extends over the cuticular plate upper surface and contacts the rootlet where it enters the plate (arrow); rat OHC. D, Vertical section of a rat OHC showing dense material in the apical meshwork along the upper edge of the plate (arrowheads). Note lighter material extending down from the mesh (white arrows) and one rootlet penetrating into the cytoplasm below the cuticular plate (black arrow). E, F, Two sets of stereopairs at low and intermediate magnification of the meshwork reconstructed from serial sections of a guinea pig OHC (cyan traces). The channels are indicated in light red in E and the basal body (normally found in the large channel on the strial side of the bundle) is shown in blue. Reconstruction shows that the meshwork around the rootlets extends in 3D so that each is ensheathed by the dense/filamentous material of the meshwork. F, Rootlets are shown as red cylinders sitting in their sheaths. Scale bars: A, D, F, 1 μm; B, C, 100 nm; E, 2 μm.

Figure 9.

Immunogold labeling for spectrin, β-actin, and γ-actin. A–E, Rat OHCs. F, G, Guinea pig OHCs. A, Spectrin is distributed throughout the cuticular plate and is evident beneath the apical membrane and along the lateral margins (arrowheads). Labeling also occurs over the rootlets (arrow). B, Spectrin labeling is evident not only in the subapical layer (arrowheads) and rootlets (arrow) but also in the ankle region (asterisk) and stereocilia (s). C, β-Actin labeling is denser over the stereocilia and rootlets than the main matrix of the cuticular plate. A labeled rootlet extends below the cuticular plate (arrow). D, As well as prominent in the rootlets (r), β-actin labeling is concentrated over the dark meshwork regions of the plate (arrows). E, The labeling density for γ-actin is similar in the stereocilia and cuticular plate. Rootlets are also labeled, one extending deeper than the cuticular plate (arrow). F, γ-Actin labeling (small particles) is also strongest over the dense patches associated with the meshwork (arrows). Large particles represent β-actin. G, γ-Actin labeling is continuous from the cortical lattice (arrowheads) into the cuticular plate (cp). Scale bars: A, C, E, 400 nm; B, D, 200 nm; F, G, 100 nm.

Figure 10.

Labeling for tropomyosin, espin, and prestin in rat OHCs. A, In horizontal sections, tropomyosin labeling is found throughout the cuticular plate but is frequently associated with rootlets (arrows). B, Rootlet labeling for tropomyosin is more apparent in vertical sections (arrows). C, Labeling for espin is in the stereocilia (s) but is not detectable in the cuticular plate or rootlets (r). Inset, Espin labeling tends to be more frequently observed near the periphery of the stereocilia. D, Prestin labeling is strong in the lateral wall but ends at the lower edge of the junctional region (arrows) and is completely absent from the apical membrane. Scale bars: A, 0.5 μm; B, D, 400 nm; C, 200 nm.

Figure 11.

A, Schematic representation of the apex of an OHC based primarily on guinea pig and rat showing how rootlets may connect to the lateral wall (arrows), as a result of the cuticular lip at the apex. The short stereociliary row is omitted for clarity. B, Schematic representation of a single rootlet (red) showing its structure. The rootlet tends to widen toward the entry point into the cell, tapering both in the stereocilium shaft and cuticular plate. The peripheral dense material coalescing with the core is also shown, coating peripheral actin filaments (green). Central actin filaments are omitted for clarity. The cell membrane is in blue and the subapical dense layer is also represented as a red lattice.