Cleaving links between stereocilia
We first confirmed that all types of links expected to be present in mature frog saccular hair cells were present in our preparations. Using transmission electron microscopy on preparations fixed without any enzyme treatment, we observed tip links (), horizontal top connectors () and ankle links (). There were also sparse links more distal than the taper region of stereocilia, which we assume are shaft connectors. If we used proteinase XXIV treatment to remove the otolithic membrane, as was done for all optical imaging experiments, the ankle links and shaft connectors were largely absent (;(Jacobs and Hudspeth, 1990
; Bashtanov et al., 2004
)). When ankle links and shaft connectors were not altogether missing, we saw what appeared to be link remnants that were disorganized (; compare to ) and in general did not span the entire distance between the stereocilia.
Figure 5 Links between stereocilia in our preparation of the bullfrog saccule; transmission electron microscopy. A, Tip link. B, Horizontal top connectors between adjacent stereocilia. C, D, Ankle links extending between adjacent stereocilia near their bases; (more ...)
To investigate which links may be responsible for the propagation of the mechanical stimuli we used biochemical treatments to selectively destroy certain links. In all imaging experiments the ankle links and shaft connectors were compromised because we used proteinase XXIV to remove the otolithic membrane. As shown in -, bundles remained together in the absence of ankle links and shaft connectors.
Tip links in dissociated bullfrog hair cells break after a brief application of the Ca2+
chelator BAPTA (Assad et al., 1991
). Using a micropipette placed about 30 μm away from a hair bundle, we applied BAPTA locally to break tip links. To test breakage, we then bath-applied FM1-43 and assessed labeling of intracellular organelles. When tip links are intact, dye enters the tips of stereocilia within seconds of dye application and passes into the cell body. After BAPTA treatment in these experiments there was no dye entry into hair cell cytoplasm for at least two minutes. We therefore concluded that the tip links were broken. Note that the tip links are thought to be composed of the same proteins as the kinocilial links that hold the kinocilium to the adjacent stereocilia (cadherin 23 and protocadherin 15; (Tsuprun et al., 2004
; Kazmierczak et al., 2007
)); not surprisingly, the kinocilium often detached from the rest of the bundle after BAPTA treatment. In these cases the stimulus probe was then attached with suction to the tallest stereocilia in order to test bundle motion.
When viewed with DIC imaging at both low and high frequencies, BAPTA-treated cells showed movement similar to non-treated cells. The measured angular deflections were similar for all stereocilia within the same column of the hair bundle, for all frequencies (). We conclude that frog saccular stereocilia are not held together by either ankle links, shaft connectors or tip links.
Figure 6 Hair bundle motion in BAPTA-treated cells. A-C, Angular displacement calculated from six cells at 1, 20 and 700 Hz respectively. Data are normalized to the displacement of the tallest stereocilium (#1) for each experiment. Stimulus displacement <500nm. (more ...)
We did not find enzymatic conditions that cleave horizontal top connectors; since they are the only links remaining they are candidates for mediating lateral adhesion. We tested this in several ways.
To further test the nature of links that might hold stereocilia together, we chose hair bundles whose morphological axis was parallel to the light path, that is, pointing straight up or down. The stimulus probe was attached by suction to stereocilia of mid-height at one edge of a bundle, and the bundle was moved perpendicular to the excitatory axis (). As can be seen in , stereocilia stayed tightly together even for sideways deflections of ~2 μm. We measured the movement of individual stereocilia of a row, and fitted the movement with the touch model, setting stereocilia heights equal. The prediction fits the observed movement well, indicating that stereocilia remain in contact even for perpendicular stimuli. Thus stereocilia are held together by a mechanism that is not oriented exclusively along a column, such as tip links, but that extends in all directions.
Figure 7 Orthogonal hair bundle stimulation. A, DIC image of a hair bundle viewed perpendicular to its excitatory axis. A stimulus probe was attached to the right edge of the bundle. B, Static stimulus of ~2 μm, perpendicular to the excitatory (more ...)
In similar experiments using interference microscopy, Kozlov et al. (Kozlov et al., 2007
) found that bullfrog saccular stereocilia remain touching when stimulated by Brownian motion, even for frequencies up to 5 kHz. Maximum deflections in these experiments were less than ~50 nm. They noted that the cuticular plate is curved, and that if the stereocilia rootlets are oriented perpendicular to the cuticular plate, it would tend to push stereocilia tips together. Such curvature could hold the tips together simply by elastic forces in the pivots, in the absence of any additional adhesion mechanism.
To test whether this might hold for larger stimuli, we first asked whether the rootlets were indeed oriented perpendicular to the curvature, by measuring rootlets from electron micrographs (). In fact, rootlets are not perpendicular, but tend to be slightly bent as they insert into the cuticular plate, by an angle that might be predicted to push the tips towards the center.
Figure 8 Angles of stereocilia rootlets. A, Transmission electron micrograph of a hair bundle where the rootlets of two stereocilia are visible (magnified in the inset). B, Method for calculating the rootlet angles. The centerline of a stereocilium (dashed) was (more ...)
We assessed the bend by measuring the angle of a rootlet relative to the angle of its stereocilium, for different stereocilia in a column (). The angle is negative for tall stereocilia, whereas it is positive for short stereocilia. If the bend in the rootlets represents a resting stress, then the force produced by the bend would push tall stereocilia in the negative direction, and would tend to push short stereocilia in the positive direction. Thus, not only does the curvature of the cuticular plate tend to orient stereocilia towards each other, but additional “prestress” in the bend of the rootlets may tend to push them together, at least to a point.
The maximum angle of prestressing is about 7° (), so if this were the sole mechanism for maintaining bundle cohesion it should work for positive deflections up to about 7° (~ 1 μm) but not beyond. We gave stimuli much larger than that to see if stereocilia separated. shows a hair bundle deflected in the positive direction by 31° (~4.5 μm) from rest. Although the stimulus probe pulled only on the bulb of the kinocilium, all the stereocilia remained in contact. The movement of individual stereocilia was measured from images (), and fitted with predictions of the geometrical model in which stereocilia are presumed to remain touching (). Even for large deflections
7°, the touch model provides a good fit.
Figure 9 Hair bundle motion in response to a large static stimulus. A, B, DIC images of a hair bundle deflected by ~ 4.5 μm or 31° in the excitatory direction. C, Peak displacements of individual stereocilia along the same column for several (more ...)
Perhaps the angles of the rootlets are not indicative of the extent of prestressing, and stereocilia are really pressed together more tightly than rootlet angles suggest. Then they might remain in contact even for large deflections. We tested this by “combing” the hair bundle with a fine glass pipet (). We first observed cohesion of stereocilia for large deflections (), then returned the bundle to the rest position. The pipet was inserted between stereocilia near the ankle region, from the side (), and then lifted up toward the tips (). We reasoned that combing in this way would disrupt any lateral links that might hold the bundle together, but would not affect prestressing of rootlets. When the bundle was again given a large positive deflection, only the taller half of the stereocilia followed the stimulus probe (). The short half remained at the rest position (compare to ), indicating that they were not intrinsically stressed towards the positive direction. Thus prestressing of stereocilia rootlets may contribute to maintaining stereocilia contact, but it seems unlikely to be the mechanism that holds them together for large deflections.
Figure 10 “Combing” away links between adjacent stereocilia. A, The tip of a fine glass probe (~ 50 nm tip diameter) was inserted through the bundle between stereocilia at their bases and then lifted toward their tips (arrows). B, The combing (more ...)
We then asked whether the links that are disrupted by combing can re-attach. We first tried pushing the taller half of the bundle back toward the short half after combing. In some cases the bundle recombined, although the apparent adhesion strength was much reduced. To further explore the reversibility of attachments we mechanically removed individual stereocilia by blotting them on a coverglass, as previously described (Shepherd et al., 1990
). Individual stereocilia were then held by a glass probe with suction and brought into contact with another stereocilium. The two stereocilia often appeared to stick to each other, especially near their tips (Supplementary Figure S1
), suggesting the presence of an intrinsic adhesive mechanism.
Thus, it seems likely that some sort of lateral link maintains stereocilia cohesion. Bundles stay together after enzymatic digestion of the ankle links and shaft connectors and after BAPTA cleavage of tip links, when the only remaining links are the horizontal top connectors. Horizontal top connectors extend among stereocilia in all directions (our own unpublished observation in frog and in chicken see (Goodyear and Richardson, 1992
; Tsuprun and Santi, 1998
)) so they could also maintain cohesion for large sideways deflections ().
Bundle cohesion in the absence of horizontal top connectors
In the chicken utricle, extrastriolar hair cell bundles have an extensive array of shaft connectors but lack morphologically distinct horizontal top connectors (Goodyear and Richardson, 1992
). We tested bundle cohesion in these hair cells from E16-E20 chick embryos, with and without treatments reported to remove all links (Bashtanov et al., 2004
). In control cells, most of the bundles moved as a unit, with shortest and tallest stereocilia moving through similar angles in response to large (>5μm) deflections (). However, even control bundles seemed more fragile than frog saccular hair cells, as about 25% of the cells tested showed some minor splay during large deflection. When the epithelia were instead treated with proteinase XXIV for 22 min followed by BAPTA, which cuts ankle links, shaft connectors and tip links (Bashtanov et al., 2004
), all of the cells splayed during deflections (). Bundle cohesion appears compromised in hair bundles that lack horizontal top connectors, especially when all other links are removed.
Figure 11 Chick utricle hair cells. Hair cells from the extrastriolar region of E16-E20 chick utricles were viewed from the side, by folding the intact epithelium and selecting hair cell bundles extending parallel to the chamber bottom. A, A control hair bundle, (more ...)
Charge and ionic interactions
Stereocilia have an extensive negatively-charged glycocalyx (Santi and Anderson, 1987
). Theoretical work predicts that if the coat charge is equally distributed within the glycocalyx or is concentrated near the outer surface, then electrostatic repulsion is able to maintain spatial separation between stereocilia (Dolgobrodov et al., 2000a
). A separate mechanism would then be required to prevent further separation and to provide an adhesive force (Flock et al., 1977
; Howard et al., 1988
; Hudspeth, 1992
We explored the effects of charge interactions on bundle adhesion by changing the ionic strength, multivalent ion concentration and pH of the bathing solution. In these experiments, the epithelia were first treated with proteinase XXIV and then with 5 mM BAPTA to remove all links except the horizontal top connectors, unless mentioned otherwise.
If the negatively charged glycocalyx produces repulsion when stereocilia membranes come in close proximity, then multivalent cations might both negate that charge and form electrostatic bridges between adjacent stereocilia. Indeed, polycations such as ruthenium red cause stereocilia to fuse near their tops under such conditions (Neugebauer and Thurm, 1987
). Consistent with this, 100 mM La3+
added to the medium caused the stereocilia to pull together more closely, and they appeared to have fused together near their tips (). When we applied displacement stimuli to such bundles, adjacent stereocilia did not slide against each other and deflection of the bundle moved the entire cell (data not shown). La3+
thus appears to lock together adjacent stereocilia, preventing sliding adhesion.
Figure 12 Ionic dependence of bundle cohesion in frog saccular hair cells. A, In the presence of 100 mM La3+. Stereocilia appear pinched together and the bundle is twisted. B, Another bundle in a medium of low ionic strength and no divalent cations. Bundle cohesion (more ...)
Neugebauer and Thurm (Neugebauer and Thurm, 1987
) found that low ionic strength solutions with only 0.2 mM Mg2+
produced a separation of hair bundles, but did not eliminate horizontal top connectors. If the horizontal top connectors are proteinaceous, it might be that the low concentration of Mg2+
was sufficient to bridge acidic residues in extracellular protein domains on adjacent stereocilia, similar to the Ca2+
stabilization of the extracellular domains in cadherin-23 (Sotomayor et al., 2010
). We tested the effect of low ionic strength by replacing all ions in the solution with D-sorbitol to maintain osmolarity (except the HEPES and NaOH needed to adjust pH; approximately 5 mM). The bundle remained cohesive even for large deflections (), ruling out an ionic bridge in adhesion but not inconsistent with a role for the horizontal top connectors.
Perhaps an extracellular protein domain has both positive and negative charges, separated by several nanometers, that attract their opposites on strands from an adjacent stereocilium. Then it may be possible to disrupt these interactions with high ionic strength. We explored this idea by increasing the concentration of NaCl in the medium from 120 mM to 5 M. The bundle remained cohesive for hypertonic solutions of less than 3M NaCl, but stereocilia tended to splay for larger concentrations of NaCl (; for the bundle shown in these panels there was no BAPTA application). High osmolarity but normal ionic strength did not produce splay. It is possible that the splay was not caused by a specific effect of high ionic strength but from a nonspecific effect of the rapid deterioration of the cells that occurred under these conditions. Consequently, we varied the pH while maintaining normal ionic strength. Bundle cohesion remained normal up to pH 10; beyond that the cells rapidly died. We then lowered the pH of the medium, by either exchanging LCR with the low pH medium or by using a pipet to puff low-pH solutions onto bundles, and looked for rapid effects that might be related to protonation of acidic residues. Conspicuous splay became evident as the pH approached 3; stereocilia separated but remained straight and pivoted at their bases (). Further exposure to such low pH caused the stereocilia to lose their stiffness and to adopt a significantly curved shape, but the splay was evident within seconds and before the stiffness was lost.