Active and passive mechanics of the mechanosensory hair-cell bundle along the tonotopic axis of the mammalian cochlea. – EARMEC

We will develop i/ an in vitro preparation of the rodent cochlea, ii/ optical tweezers that will allow for fast mechanical stimulation of hair cells with laser light, iii/ a method allowing to quickly change the caclium concentration near a hair bundle, iv/ mutant mice allowing to interfere specifically with two myosin molecular motors in the hair bundle, and v/ a technique of fast calcium imaging.

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Hearing is initiated by specialized mechanosensory “hair cells” that transduce sound-evoked mechanical vibrations into electrical signals. Hair cells do not only function as cellular microphones, they also produce mechanical work to actively amplify their responsiveness to faint sound stimuli. As the result of active hair-cell motility, the ear achieves exquisite sensitivity, sharp frequency selectivity and can operate over six orders of magnitude of sound-pressure levels. In addition, our ability to communicate and appreciate music relies on acute frequency discrimination over a broad range of sound frequencies. Humans are sensitive to sounds with frequencies that vary by one-thousand fold, from 20 Hz to 20 kHz. Some other mammals like bats and cetaceans can hear frequencies up to 100 kHz. These characteristics impose stringent constraints on the mechanical properties of the hair cells, active (force generation) and passive (stiffness, mass, and drag), because these properties control how well these cells will vibrate and in turn respond to incoming sound signals. The cochlea, the auditory organ of the mammalian ear, is endowed with several thousand hair cells (16,000 in humans) that are regularly distributed along a tonotopic axis. Along this axis, the hair cells are each tuned to respond most sensitively at a characteristic frequency that decreases exponentially from base to apex of the cochlea. The cellular and molecular mechanism that specifies at which stimulus frequency a given hair cell ought to respond and amplify most effectively is largely unknown and constitutes a central question of auditory physiology.

The hair bundle, the mechano-sensory organelle that protrudes from the apical surface of each hair cell, is thought to play a key role. The hair bundle indeed displays morphofunctional characteristics that vary all along the tonotopic axis of cochlea. It has also been shown to produce active movements that may both enhance and filter the input to the hair cell. The hair-bundle structure and motile properties must be tightly controled by the bundle’s molecular constituents. The elucidation of the molecular bases of deafness has in recent years provided dozens of entry points in the cochlear structure, especially within the hair bundle. The emerging molecular networks, however, need to be linked to the mechano-sensitivity of the hair bundle bridge hair-bundle biophysics and the underlying molecular players.

In this project, we aim at elucidating the implication of the hair bundle in setting the characteristic frequency of a sensory hair cell. We propose to combine fast stimulation of single hair bundles with genetically engineered mouse mutants to characterize passive and active mechanical properties of the hair-cell bundle along the tonotopic axis of the rodent cochlea and test thes effects of two myosins on these properties. Because the calcium component of the mechano-to-electrical transduction current is known to be essential for the generation of active hair-bundle movements, we will use fast imaging to localize the site(s) of Ca2+ entry in the hair bundle and confront our observations to conflicting reports from the literature. We plan to investigate (i) passive and active components to the stiffness of a hair-cell bundle, (ii) the resting tip-link tension in the hair bundle, (iii) the effects of a local variation of the Ca2+ concentration on hair-bundle mechanics, and (iv) the ability of a hair bundle to respond actively to step forces and to oscillate spontaneously. Finally, the information produced by this study will be integrated in a theoretical model of active hair-bundle mechanics that should clarify the contribution of the hair bundle on the dazzling sensitivity and frequency selectivity that is evinced by mammalian hearing.