Development of an improved inhibitor of Lats kinases to promote regeneration of mammalian organs.

The Hippo signaling pathway acts as a brake on regeneration in many tissues. This cascade of kinases culminates in the phosphorylation of the transcriptional cofactors Yap and Taz, whose concentration in the nucleus consequently remains low. Various types of cellular signals can reduce phosphorylation, however, resulting in the accumulation of Yap and Taz in the nucleus and subsequently in mitosis. We earlier identified a small molecule, TRULI, that blocks the final kinases in the pathway, Lats1 and Lats2, and thus elicits proliferation of several cell types that are ordinarily postmitotic and aids regeneration in mammals. In the present study, we present the results of chemical modification of the original compound and demonstrate that a derivative, TDI-011536, is an effective blocker of Lats kinases in vitro at nanomolar concentrations. The compound fosters extensive proliferation in retinal organoids derived from human induced pluripotent stem cells. Intraperitoneal administration of the substance to mice suppresses Yap phosphorylation for several hours and induces transcriptional activation of Yap target genes in the heart, liver, and skin. Moreover, the compound initiates the proliferation of cardiomyocytes in adult mice following cardiac cryolesions. After further chemical refinement, related compounds might prove useful in protective and regenerative therapies.

Fast recovery of disrupted tip links induced by mechanical displacement of hair bundles.

Hearing and balance rely on the capacity of mechanically sensitive hair bundles to transduce vibrations into electrical signals that are forwarded to the brain. Hair bundles possess tip links that interconnect the mechanosensitive stereocilia and convey force to the transduction channels. A dimer of dimers, each of these links comprises two molecules of protocadherin 15 (PCDH15) joined to two of cadherin 23 (CDH23). The "handshake" that conjoins the four molecules can be disrupted in vivo by intense stimulation and in vitro by exposure to Ca2+ chelators. Using hair bundles from the rat's cochlea and the bullfrog's sacculus, we observed that extensive recovery of mechanoelectrical transduction, hair bundle stiffness, and spontaneous bundle oscillation can occur within seconds after Ca2+ chelation, especially if hair bundles are deflected toward their short edges. Investigating the phenomenon in a two-compartment ionic environment that mimics natural conditions, we combined iontophoretic application of a Ca2+ chelator to selectively disrupt the tip links of individual frog hair bundles with displacement clamping to control hair bundle motion and measure forces. Our observations suggest that, after the normal Ca2+ concentration has been restored, mechanical stimulation facilitates the reconstitution of functional tip links.

Modeling Active Non-Markovian Oscillations.

Modeling noisy oscillations of active systems is one of the current challenges in physics and biology. Because the physical mechanisms of such processes are often difficult to identify, we propose a linear stochastic model driven by a non-Markovian bistable noise that is capable of generating self-sustained periodic oscillation. We derive analytical predictions for most relevant dynamical and thermodynamic properties of the model. This minimal model turns out to describe accurately bistablelike oscillatory motion of hair bundles in bullfrog sacculus, extracted from experimental data. Based on and in agreement with these data, we estimate the power required to sustain such active oscillations to be of the order of 100 k_{B}T per oscillation cycle.

Elasticity of individual protocadherin 15 molecules implicates tip links as the gating springs for hearing.

Hair cells, the sensory receptors of the inner ear, respond to mechanical forces originating from sounds and accelerations. An essential feature of each hair cell is an array of filamentous tip links, consisting of the proteins protocadherin 15 (PCDH15) and cadherin 23 (CDH23), whose tension is thought to directly gate the cell's transduction channels. These links are considered far too stiff to represent the gating springs that convert hair bundle displacement into forces capable of opening the channels, and no mechanism has been suggested through which tip-link stiffness could be varied to accommodate hair cells of distinct frequency sensitivity in different receptor organs and animals. Consequently, the gating spring's identity and mechanism of operation remain central questions in sensory neuroscience. Using a high-precision optical trap, we show that an individual monomer of PCDH15 acts as an entropic spring that is much softer than its enthalpic stiffness alone would suggest. This low stiffness implies that the protein is a significant part of the gating spring that controls a hair cell's transduction channels. The tip link's entropic nature then allows for stiffness control through modulation of its tension. We find that a PCDH15 molecule is unstable under tension and exhibits a rich variety of reversible unfolding events that are augmented when the Ca2+ concentration is reduced to physiological levels. Therefore, tip link tension and Ca2+ concentration are likely parameters through which nature tunes a gating spring's mechanical properties.

Daple coordinates organ-wide and cell-intrinsic polarity to pattern inner-ear hair bundles.

The establishment of planar polarization by mammalian cells necessitates the integration of diverse signaling pathways. In the inner ear, at least two systems regulate the planar polarity of sensory hair bundles. The core planar cell polarity (PCP) proteins coordinate the orientations of hair cells across the epithelial plane. The cell-intrinsic patterning of hair bundles is implemented independently by the G protein complex classically known for orienting the mitotic spindle. Although the primary cilium also participates in each of these pathways, its role and the integration of the two systems are poorly understood. We show that Dishevelled-associating protein with a high frequency of leucine residues (Daple) interacts with PCP and cell-intrinsic signals. Regulated by the cell-intrinsic pathway, Daple is required to maintain the polarized distribution of the core PCP protein Dishevelled and to position the primary cilium at the abneural edge of the apical surface. Our results suggest that the primary cilium or an associated structure influences the domain of cell-intrinsic signals that shape the hair bundle. Daple is therefore essential to orient and pattern sensory hair bundles.

Homeostatic enhancement of sensory transduction.

Our sense of hearing boasts exquisite sensitivity, precise frequency discrimination, and a broad dynamic range. Experiments and modeling imply, however, that the auditory system achieves this performance for only a narrow range of parameter values. Small changes in these values could compromise hair cells' ability to detect stimuli. We propose that, rather than exerting tight control over parameters, the auditory system uses a homeostatic mechanism that increases the robustness of its operation to variation in parameter values. To slowly adjust the response to sinusoidal stimulation, the homeostatic mechanism feeds back a rectified version of the hair bundle's displacement to its adaptation process. When homeostasis is enforced, the range of parameter values for which the sensitivity, tuning sharpness, and dynamic range exceed specified thresholds can increase by more than an order of magnitude. Signatures in the hair cell's behavior provide a means to determine through experiment whether such a mechanism operates in the auditory system. Robustness of function through homeostasis may be ensured in any system through mechanisms similar to those that we describe here.

Bilateral Spontaneous Otoacoustic Emissions Show Coupling between Active Oscillators in the Two Ears.

Spontaneous otoacoustic emissions (SOAEs) are weak sounds that emanate from the ears of tetrapods in the absence of acoustic stimulation. These emissions are an epiphenomenon of the inner ear's active process, which enhances the auditory system's sensitivity to weak sounds, but their mechanism of production remains a matter of debate. We recorded SOAEs simultaneously from the two ears of the tokay gecko and found that binaural emissions could be strongly correlated: some emissions occurred at the same frequency in both ears and were highly synchronized. Suppression of the emissions in one ear often changed the amplitude or shifted the frequency of emissions in the other. Decreasing the frequency of emissions from one ear by lowering its temperature usually reduced the frequency of the contralateral emissions. To understand the relationship between binaural SOAEs, we developed a mathematical model of the eardrums as noisy nonlinear oscillators coupled by the air within an animal's mouth. By according with the model, the results indicate that some SOAEs are generated bilaterally through acoustic coupling across the oral cavity. The model predicts that sound localization through the acoustic coupling between ears is influenced by the active processes of both ears.

Integrating the active process of hair cells with cochlear function.

Uniquely among human senses, hearing is not simply a passive response to stimulation. Our auditory system is instead enhanced by an active process in cochlear hair cells that amplifies acoustic signals several hundred-fold, sharpens frequency selectivity and broadens the ear's dynamic range. Active motility of the mechanoreceptive hair bundles underlies the active process in amphibians and some reptiles; in mammals, this mechanism operates in conjunction with prestin-based somatic motility. Both individual hair bundles and the cochlea as a whole operate near a dynamical instability, the Hopf bifurcation, which accounts for the cardinal features of the active process.

Chemomechanical regulation of myosin Ic cross-bridges: Deducing the elastic properties of an ensemble from single-molecule mechanisms.

Myosin Ic is thought to be the principal constituent of the motor that adjusts mechanical responsiveness during adaptation to prolonged stimuli by hair cells, the sensory receptors of the inner ear. In this context myosin molecules operate neither as filaments, as occurs in muscles, nor as single or few molecules, as characterizes intracellular transport. Instead, myosin Ic molecules occur in a complex cluster in which they may exhibit cooperative properties. To better understand the motor's remarkable function, we introduce a theoretical description of myosin Ic's chemomechanical cycle based on experimental data from recent single-molecule studies. The cycle consists of distinct chemical states that the myosin molecule stochastically occupies. We explicitly calculate the probabilities of the occupancy of these states and show their dependence on the external force, the availability of actin, and the nucleotide concentrations as required by thermodynamic constraints. This analysis highlights that the strong binding of myosin Ic to actin is dominated by the ADP state for small external forces and by the ATP state for large forces. Our approach shows how specific parameter values of the chemomechanical cycle for myosin Ic result in behaviors distinct from those of other members of the myosin family. Integrating this single-molecule cycle into a simplified ensemble description, we predict that the average number of bound myosin heads is regulated by the external force and nucleotide concentrations. The elastic properties of such an ensemble are determined by the average number of myosin cross-bridges. Changing the binding probabilities and myosin's stiffness under a constant force results in a mechanical relaxation which is large enough to account for fast adaptation in hair cells.

SoxC transcription factors are essential for the development of the inner ear.

Hair cells, the mechanosensory receptors of the inner ear, underlie the senses of hearing and balance. Adult mammals cannot adequately replenish lost hair cells, whose loss often results in deafness or balance disorders. To determine the molecular basis of this deficiency, we investigated the development of a murine vestibular organ, the utricle. Here we show that two members of the SoxC family of transcription factors, Sox4 and Sox11, are down-regulated after the epoch of hair cell development. Conditional ablation of SoxC genes in vivo results in stunted sensory organs of the inner ear and loss of hair cells. Enhanced expression of SoxC genes in vitro conversely restores supporting cell proliferation and the production of new hair cells in adult sensory epithelia. These results imply that SoxC genes govern hair cell production and thus advance these genes as targets for the restoration of hearing and balance.

Control of a hair bundle's mechanosensory function by its mechanical load.

Hair cells, the sensory receptors of the internal ear, subserve different functions in various receptor organs: they detect oscillatory stimuli in the auditory system, but transduce constant and step stimuli in the vestibular and lateral-line systems. We show that a hair cell's function can be controlled experimentally by adjusting its mechanical load. By making bundles from a single organ operate as any of four distinct types of signal detector, we demonstrate that altering only a few key parameters can fundamentally change a sensory cell's role. The motions of a single hair bundle can resemble those of a bundle from the amphibian vestibular system, the reptilian auditory system, or the mammalian auditory system, demonstrating an essential similarity of bundles across species and receptor organs.

Forces between clustered stereocilia minimize friction in the ear on a subnanometre scale.

The detection of sound begins when energy derived from an acoustic stimulus deflects the hair bundles on top of hair cells. As hair bundles move, the viscous friction between stereocilia and the surrounding liquid poses a fundamental physical challenge to the ear's high sensitivity and sharp frequency selectivity. Part of the solution to this problem lies in the active process that uses energy for frequency-selective sound amplification. Here we demonstrate that a complementary part of the solution involves the fluid-structure interaction between the liquid within the hair bundle and the stereocilia. Using force measurement on a dynamically scaled model, finite-element analysis, analytical estimation of hydrodynamic forces, stochastic simulation and high-resolution interferometric measurement of hair bundles, we characterize the origin and magnitude of the forces between individual stereocilia during small hair-bundle deflections. We find that the close apposition of stereocilia effectively immobilizes the liquid between them, which reduces the drag and suppresses the relative squeezing but not the sliding mode of stereociliary motion. The obliquely oriented tip links couple the mechanotransduction channels to this least dissipative coherent mode, whereas the elastic horizontal top connectors that stabilize the structure further reduce the drag. As measured from the distortion products associated with channel gating at physiological stimulation amplitudes of tens of nanometres, the balance of viscous and elastic forces in a hair bundle permits a relative mode of motion between adjacent stereocilia that encompasses only a fraction of a nanometre. A combination of high-resolution experiments and detailed numerical modelling of fluid-structure interactions reveals the physical principles behind the basic structural features of hair bundles and shows quantitatively how these organelles are adapted to the needs of sensitive mechanotransduction.

Reaction-diffusion model of hair-bundle morphogenesis.

The hair bundle, an apical specialization of the hair cell composed of several rows of regularly organized stereocilia and a kinocilium, is essential for mechanotransduction in the ear. Its precise organization allows the hair bundle to convert mechanical stimuli to electrical signals; mutations that alter the bundle's morphology often cause deafness. However, little is known about the proteins involved in the process of morphogenesis and how the structure of the bundle arises through interactions between these molecules. We present a mathematical model based on simple reaction-diffusion mechanisms that can reproduce the shape and organization of the hair bundle. This model suggests that the boundary of the cell and the kinocilium act as signaling centers that establish the bundle's shape. The interaction of two proteins forms a hexagonal Turing pattern--a periodic modulation of the concentrations of the morphogens, sustained by local activation and long-range inhibition of the reactants--that sets a blueprint for the location of the stereocilia. Finally we use this model to predict how different alterations to the system might impact the shape and organization of the hair bundle.

Dynamic gene expression by putative hair-cell progenitors during regeneration in the zebrafish lateral line.

Hearing loss is most commonly caused by the destruction of mechanosensory hair cells in the ear. This condition is usually permanent: Despite the presence of putative hair-cell progenitors in the cochlea, hair cells are not naturally replenished in adult mammals. Unlike those of the mammalian ear, the progenitor cells of nonmammalian vertebrates can regenerate hair cells throughout life. The basis of this difference remains largely unexplored but may lie in molecular dissimilarities that affect how progenitors respond to hair-cell death. To approach this issue, we analyzed gene expression in hair-cell progenitors of the lateral-line system. We developed a transgenic line of zebrafish that expresses a red fluorescent protein in the presumptive hair-cell progenitors known as mantle cells. Fluorescence-activated cell sorting from the skins of transgenic larvae, followed by microarray-based expression analysis, revealed a constellation of transcripts that are specifically enriched in these cells. Gene expression analysis after hair-cell ablation uncovered a cohort of genes that are differentially regulated early in regeneration, suggesting possible roles in the response of progenitors to hair-cell death. These results provide a resource for studying hair-cell regeneration and the biology of sensory progenitor cells.

Identification of Bifurcations from Observations of Noisy Biological Oscillators.

Hair bundles are biological oscillators that actively transduce mechanical stimuli into electrical signals in the auditory, vestibular, and lateral-line systems of vertebrates. A bundle's function can be explained in part by its operation near a particular type of bifurcation, a qualitative change in behavior. By operating near different varieties of bifurcation, the bundle responds best to disparate classes of stimuli. We show how to determine the identity of and proximity to distinct bifurcations despite the presence of substantial environmental noise. Using an improved mechanical-load clamp to coerce a hair bundle to traverse different bifurcations, we find that a bundle operates within at least two functional regimes. When coupled to a high-stiffness load, a bundle functions near a supercritical Hopf bifurcation, in which case it responds best to sinusoidal stimuli such as those detected by an auditory organ. When the load stiffness is low, a bundle instead resides close to a subcritical Hopf bifurcation and achieves a graded frequency response-a continuous change in the rate, but not the amplitude, of spiking in response to changes in the offset force-a behavior that is useful in a vestibular organ. The mechanical load in vivo might therefore control a hair bundle's responsiveness for effective operation in a particular receptor organ. Our results provide direct experimental evidence for the existence of distinct bifurcations associated with a noisy biological oscillator, and demonstrate a general strategy for bifurcation analysis based on observations of any noisy system.

Effects of cochlear loading on the motility of active outer hair cells.

Outer hair cells (OHCs) power the amplification of sound-induced vibrations in the mammalian inner ear through an active process that involves hair-bundle motility and somatic motility. It is unclear, though, how either mechanism can be effective at high frequencies, especially when OHCs are mechanically loaded by other structures in the cochlea. We address this issue by developing a model of an active OHC on the basis of observations from isolated cells, then we use the model to predict the response of an active OHC in the intact cochlea. We find that active hair-bundle motility amplifies the receptor potential that drives somatic motility. Inertial loading of a hair bundle by the tectorial membrane reduces the bundle's reactive load, allowing the OHC's active motility to influence the motion of the cochlear partition. The system exhibits enhanced sensitivity and tuning only when it operates near a dynamical instability, a Hopf bifurcation. This analysis clarifies the roles of cochlear structures and shows how the two mechanisms of motility function synergistically to create the cochlear amplifier. The results suggest that somatic motility evolved to enhance a preexisting amplifier based on active hair-bundle motility, thus allowing mammals to hear high-frequency sounds.

Anomalous Brownian motion discloses viscoelasticity in the ear's mechanoelectrical-transduction apparatus.

The ear detects sounds so faint that they produce only atomic-scale displacements in the mechanoelectrical transducer, yet thermal noise causes fluctuations larger by an order of magnitude. Explaining how hearing can operate when the magnitude of the noise greatly exceeds that of the signal requires an understanding both of the transducer's micromechanics and of the associated noise. Using microrheology, we characterize the statistics of this noise; exploiting the fluctuation-dissipation theorem, we determine the associated micromechanics. The statistics reveal unusual Brownian motion in which the mean square displacement increases as a fractional power of time, indicating that the mechanisms governing energy dissipation are related to those of energy storage. This anomalous scaling contradicts the canonical model of mechanoelectrical transduction, but the results can be explained if the micromechanics incorporates viscoelasticity, a salient characteristic of biopolymers. We amend the canonical model and demonstrate several consequences of viscoelasticity for sensory coding.

The diverse effects of mechanical loading on active hair bundles.

Hair cells in the auditory, vestibular, and lateral-line systems of vertebrates receive inputs through a remarkable variety of accessory structures that impose complex mechanical loads on the mechanoreceptive hair bundles. Although the physiological and morphological properties of the hair bundles in each organ are specialized for detecting the relevant inputs, we propose that the mechanical load on the bundles also adjusts their responsiveness to external signals. We use a parsimonious description of active hair-bundle motility to show how the mechanical environment can regulate a bundle's innate behavior and response to input. We find that an unloaded hair bundle can behave very differently from one subjected to a mechanical load. Depending on how it is loaded, a hair bundle can function as a switch, active oscillator, quiescent resonator, or low-pass filter. Moreover, a bundle displays a sharply tuned, nonlinear, and sensitive response for some loading conditions and an untuned or weakly tuned, linear, and insensitive response under other circumstances. Our simple characterization of active hair-bundle motility explains qualitatively most of the observed features of bundle motion from different organs and organisms. The predictions stemming from this description provide insight into the operation of hair bundles in a variety of contexts.

Contribution of active hair-bundle motility to nonlinear amplification in the mammalian cochlea.

The cochlea's high sensitivity stems from the active process of outer hair cells, which possess two force-generating mechanisms: active hair-bundle motility elicited by Ca(2+) influx and somatic motility mediated by the voltage-sensitive protein prestin. Although interference with prestin has demonstrated a role for somatic motility in the active process, it remains unclear whether hair-bundle motility contributes in vivo. We selectively perturbed the two mechanisms by infusing substances into the endolymph or perilymph of the chinchilla's cochlea and then used scanning laser interferometry to measure vibrations of the basilar membrane. Blocking somatic motility, damaging the tip links of hair bundles, or depolarizing hair cells eliminated amplification. While reducing amplification to a lesser degree, pharmacological perturbation of active hair-bundle motility diminished or eliminated the nonlinear compression underlying the broad dynamic range associated with normal hearing. The results suggest that active hair-bundle motility plays a significant role in the amplification and compressive nonlinearity of the cochlea.

The physics of hearing: fluid mechanics and the active process of the inner ear.

Most sounds of interest consist of complex, time-dependent admixtures of tones of diverse frequencies and variable amplitudes. To detect and process these signals, the ear employs a highly nonlinear, adaptive, real-time spectral analyzer: the cochlea. Sound excites vibration of the eardrum and the three miniscule bones of the middle ear, the last of which acts as a piston to initiate oscillatory pressure changes within the liquid-filled chambers of the cochlea. The basilar membrane, an elastic band spiraling along the cochlea between two of these chambers, responds to these pressures by conducting a largely independent traveling wave for each frequency component of the input. Because the basilar membrane is graded in mass and stiffness along its length, however, each traveling wave grows in magnitude and decreases in wavelength until it peaks at a specific, frequency-dependent position: low frequencies propagate to the cochlear apex, whereas high frequencies culminate at the base. The oscillations of the basilar membrane deflect hair bundles, the mechanically sensitive organelles of the ear's sensory receptors, the hair cells. As mechanically sensitive ion channels open and close, each hair cell responds with an electrical signal that is chemically transmitted to an afferent nerve fiber and thence into the brain. In addition to transducing mechanical inputs, hair cells amplify them by two means. Channel gating endows a hair bundle with negative stiffness, an instability that interacts with the motor protein myosin-1c to produce a mechanical amplifier and oscillator. Acting through the piezoelectric membrane protein prestin, electrical responses also cause outer hair cells to elongate and shorten, thus pumping energy into the basilar membrane's movements. The two forms of motility constitute an active process that amplifies mechanical inputs, sharpens frequency discrimination, and confers a compressive nonlinearity on responsiveness. These features arise because the active process operates near a Hopf bifurcation, the generic properties of which explain several key features of hearing. Moreover, when the gain of the active process rises sufficiently in ultraquiet circumstances, the system traverses the bifurcation and even a normal ear actually emits sound. The remarkable properties of hearing thus stem from the propagation of traveling waves on a nonlinear and excitable medium.