Direct mechanical stimulation of tip links in hair cells through DNA tethers

Abstract

Mechanoelectrical transduction by hair cells commences with hair-bundle deflection, which is postulated to tense filamentous tip links connected to transduction channels. Because direct mechanical stimulation of tip links has not been experimentally possible, this hypothesis has not been tested. We have engineered DNA tethers that link superparamagnetic beads to tip links and exert mechanical forces on the links when exposed to a magnetic-field gradient. By pulling directly on tip links of the bullfrog's sacculus we have evoked transduction currents from hair cells, confirming the hypothesis that tension in the tip links opens transduction channels. This demonstration of direct mechanical access to tip links additionally lays a foundation for experiments probing the mechanics of individual channels.

Keywords: auditory system; biophysics; bullfrog; mechanoelectrical transduction; neuroscience; structural biology; vestibular system.

Figure 1.. Experimental configuration and control experiments.

(A) In diagrams of a hair cell at rest (top) and during excitatory stimulation (bottom), the magnified images show how hair-bundle deflection is thought to raise the tension in tip links and thus open transduction channels. (B) A diagram, not to scale, portrays the molecular assembly that tethers a superparamagnetic bead to a tip link. (C) In overlaid fluorescence and differential-interference-contrast images of the apical surface of a bullfrog's sacculus, anti-CDH23 immunolabels the tops of hair bundles. (D) Labeling is absent under otherwise identical conditions when the primary antibody is omitted. (E) A differential-interference-contrast micrograph of a sacculus shows clusters of DNA-tethered superparamagnetic beads atop many hair bundles. (F) No beads are present in a control preparation lacking the primary antibody. DOI: http://dx.doi.org/10.7554/eLife.16041.003

Figure 2.. Electrical responses to magnetic stimulation.

(A) The diagram shows a sacculus mounted in a two-compartment chamber beneath an electromagnet. The otolithic membrane has been removed and superparamagnetic beads (red dots) are attached to the tip links through DNA tethers. (B) Brief magnetic stimuli of opposite polarities, which are denoted by the voltages applied to the magnet, elicit transepithelial electrical responses. Exposure of the preparation to 1 mM gentamicin blocks these physiological responses, leaving only artifacts that are eliminated by averaging the signals obtained from stimuli of opposite polarity. (C) Longer stimuli demonstrate the persistence of transduction currents without the extensive adaptation observed during conventional stimulation. The stimulus traces shown produced the largest response; successively smaller responses were generated by lowering the voltage supplied to the magnet in steps of 0.2 V. (D) To eliminate the possibility that magnetic stimulation pulls hair bundles in the excitatory direction, most of the beads can be removed and the preparation tilted by 10°. (E) Brown superparamagnetic beads initially cover the apical surface of a mounted sacculus (left). Scraping away most of the beads (center) leaves only the beads attached to hair bundles of similar orientations (arrowheads), sensitive to stimulation away from the saccular nerve (N). Further manipulation removes nearly all the beads (right). The dashed line circumscribes the region in which hair cells occur. (F) Although smaller than the control response (top), an electrical response nevertheless persists after removal of most beads (middle). With the beads localized to one region with similar hair-bundle orientations, magnetic force pulls those bundles upward and toward their short edges. Removing the remaining beads eliminates the response (bottom). The calibration bars in (C) apply to this panel as well. (G) Addition to the artificial perilymph in the top chamber of 1 mM EGTA, a Ca²⁺ chelator that dissociates tip links, abolished the electrical response. The calibration bars in (C) apply to this panel as well. (H) When a superparamagnetic bead is tethered by DNA to a glass surface through DNA, thermal motion causes lateral deflections that are partially suppressed by the magnetic force F. DOI: http://dx.doi.org/10.7554/eLife.16041.004