Hair Bundle Research Articles

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish.

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The Transfer Characteristics of Hair Cells Encoding Mechanical Stimuli in the Lateral Line of Zebrafish

Hair cells transmit mechanical information by converting deflection of the hair bundle into synaptic release of glutamate. We have investigated this process in the lateral line of larval zebrafish (male and female) to understand how stimuli are encoded within a neuromast. Using multiphoton microscopy in vivo, we imaged synaptic release of glutamate using the reporter iGluSnFR as well as deflections of the cupula. We found that the neuromast is composed of a functionally diverse population of hair cells. Half the hair cells signaled cupula motion in both directions from rest, either by increasing glutamate release in response to a deflection in the positive direction or by reducing release in the negative direction. The relationship between cupula deflection and glutamate release demonstrated maximum sensitivity at displacements of just ∼40 nm in the positive direction. The remaining hair cells only signaled motion in one direction and were less sensitive, extending the operating range of the neuromast beyond 1 μm. Adaptation of the synaptic output was also heterogeneous, with some hair cells generating sustained glutamate release in response to a steady deflection of the cupula and others generating transient outputs. Finally, a distinct signal encoded a return of the cupula to rest: a large and transient burst of glutamate release from hair cells unresponsive to the initial stimulus. A population of hair cells with these different sensitivities, operating ranges, and adaptive properties will allow the neuromast to encode weak stimuli while maintaining the dynamic range to signal the amplitude and duration of stronger deflections.SIGNIFICANCE STATEMENT Hair cells transmit information about mechanical stimuli by converting very small deflections of their hair bundle into changes in the release of the neurotransmitter glutamate. We have measured this input/output relation in the live fish using a fluorescent protein and find that different hair cells vary in their mechanical sensitivity and the time course of their response. These variations will allow the fish to sense the timing and duration of both very weak stimuli (∼40 nm deflections) and strong stimuli (∼1 μm), underlying the ability of the fish to avoid predators and maintain its body position in flowing water.

Generation of mixed-mode stochastic oscillations in a hair bundle model

We consider a mathematical model of spontaneous mechanical oscillations of hair bundles in bullfrog saccular hair cells. This two-dimensional model describes dynamics of the positions of the bundle tip and molecular motors. The main control parameters of this model are the calcium feedback strength and the maximal force of molecular motors. The dynamics of hair bundles is subject to the random disturbances. We study noise-induced oscillations in the parametric zone where the equilibria are the only attractors of the system. We analyze the probabilistic mechanism of the generation of mixed-mode stochastic oscillations with the help of the stochastic sensitivity functions and confidence domains. The constructive abilities of this mathematical technique in the analysis of the stochastic effects in the hair bundle model are demonstrated.

Active Biomechanics of Sensory Hair Bundles.

During the detection of sound, hair bundles perform a crucial step by responding to mechanical deflections and converting them into changes in electrical potential that subsequently lead to the release of neurotransmitter. The sensory hair bundle response is characterized by an essential nonlinearity and an energy-consuming amplification of the incoming sound. The active response has been shown to enhance the hair bundle's sensitivity and frequency selectivity of detection. The biological phenomena shown by the bundle have been extensively studied in vitro, allowing comparisons to behaviors observed in vivo. The experimental observations have been well explained by numerical simulations, which describe the cellular mechanisms operant within the bundle, as well as by more sparse theoretical models, based on dynamical systems theory.

Grxcr1 Promotes Hair Bundle Development by Destabilizing the Physical Interaction between Harmonin and Sans Usher Syndrome Proteins

SUMMARYMorphogenesis and mechanoelectrical transduction of the hair cell mechanoreceptor depend on the correct assembly of Usher syndrome (USH) proteins into highly organized macromolecular complexes. Defects in these proteins lead to deafness and vestibular areflexia in USH patients. Mutations in a non-USH protein, glutaredoxin domain-containing cysteine-rich 1 (GRXCR1), cause non-syndromic sensorineural deafness. To understand the deglutathionylating enzyme function of GRXCR1 in deafness, we generated two grxcr1 zebrafish mutant alleles. We found that hair bundles are thinner in homozygous grxcr1 mutants, similar to the USH1 mutants ush1c (Harmonin) and ush1ga (Sans). In vitro assays showed that glutathionylation promotes the interaction between Ush1c and Ush1ga and that Grxcr1 regulates mechanoreceptor development by preventing physical interaction between these proteins without affecting the assembly of another USH1 protein complex, the Ush1c-Cadherin23-Myosin7aa tripartite complex. By elucidating the molecular mechanism through which Grxcr1 functions, we also identify a mechanism that dynamically regulates the formation of Usher protein complexes.

Deletion of Kncn Does Not Affect Kinocilium and Stereocilia Bundle Morphogenesis and Mechanotransduction in Cochlear Hair Cells.

Auditory hair cells possess stunning cilia structure that composes of a bundle of stereocilia for mechano-electrical transduction and a single kinocilium for guiding the polarity of hair bundle towards maturation. However, the molecules underlying kinocilium function have not yet been fully understood. Hence, the proteins involved in hair bundle development and function are of a large interest. From a fine microarray analysis, we found that kinocilin (Kncn) was enriched in hair cell specific expression profile. Consistently, it has been reported that KNCN was a protein mainly located in the kinocilium of hair cells in the inner ear. However, the hypothesis that KNCN is a kinocilium protein has not been validated in mice with Kncn gene perturbed. In this study, we generated Kncn knockout mouse lines by CRISPR/Cas9 technique and further examined the morphology and function of cochlear hair cells. Our results showed that there was no obvious hearing loss in the knockout mice, determined by audiometry. Histological study demonstrated that the inner ear and hair cell structure were intact. Especially, there was no deficit of mechanotransduction (MET) in cochlear outer hair cells (OHCs). In summary, our work suggests that KNCN is not essential for kinocilium-oriented hair bundle function in cochlear hair cells.

Nonlinear calcium ion waves along actin filaments control active hair–bundle motility

Calcium ions (Ca2+) tune and control numerous diverse aspects of cochlear and vestibular physiological processes. This paper is focused on the Ca2+ control of mechanotransduction in sensory hair cells in the context of polyelectrolyte properties of actin filaments within the hair–bundles of inner ear. These actin filaments appear to serve as efficient pathways for the flow of Ca2+ ions inside stereocilia. We showed how this can be utilized for tuning of force–generating myosin motors. In an established model, we unified the Ca2+ nonlinear dynamics involved in the control of myosin adaptation motors with mechanical displacements of hair–bundles. The model shows that the characteristic time scales fit reasonably well with the available experimental data for spontaneous oscillations in the inner ear. This scenario promises to fill a gap in our understanding of the role of Ca2+ ions in the regulation of processes in the auditory cells of the inner ear.

Deletion of Mtu1 (Trmu) in zebrafish revealed the essential role of tRNA modification in mitochondrial biogenesis and hearing function.

Mtu1(Trmu) is a highly conserved tRNA modifying enzyme responsible for the biosynthesis of τm5s2U at the wobble position of tRNAGln, tRNAGlu and tRNALys. Our previous investigations showed that MTU1 mutation modulated the phenotypic manifestation of deafness-associated mitochondrial 12S rRNA mutation. However, the pathophysiology of MTU1 deficiency remains poorly understood. Using the mtu1 knock-out zebrafish generated by CRISPR/Cas9 system, we demonstrated the abolished 2-thiouridine modification of U34 of mitochondrial tRNALys, tRNAGlu and tRNAGln in the mtu1 knock-out zebrafish. The elimination of this post-transcriptional modification mediated mitochondrial tRNA metabolisms, causing the global decreases in the levels of mitochondrial tRNAs. The aberrant mitochondrial tRNA metabolisms led to the impairment of mitochondrial translation, respiratory deficiencies and reductions of mitochondrial ATP production. These mitochondria dysfunctions caused the defects in hearing organs. Strikingly, mtu1−/– mutant zebrafish displayed the abnormal startle response and swimming behaviors, significant decreases in the sizes of saccular otolith and numbers of hair cells in the auditory and vestibular organs. Furthermore, mtu1−/– mutant zebrafish exhibited the significant reductions in the hair bundle densities in utricle, saccule and lagena. Therefore, our findings may provide new insights into the pathophysiology of deafness, which was manifested by the deficient modifications at wobble position of mitochondrial tRNAs.

SELECTION AND GENETIC CHARACTERISTICS OF FOXES AT CAGE BREEDING

SELECTION AND GENETIC CHARACTERISTICS OF FOXES AT CAGE BREEDING

FGFR1-mediated protocadherin-15 loading mediates cargo specificity during intraflagellar transport in inner ear hair-cell kinocilia

The mechanosensory hair cells of the inner ear are required for hearing and balance and have a distinctive apical structure, the hair bundle, that converts mechanical stimuli into electrical signals. This structure comprises a single cilium, the kinocilium, lying adjacent to an ensemble of actin-based projections known as stereocilia. Hair bundle polarity depends on kinociliary protocadherin-15 (Pcdh15) localization. Protocadherin-15 is found only in hair-cell kinocilia, and is not localized to the primary cilia of adjacent supporting cells. Thus, Pcdh15 must be specifically targeted and trafficked into the hair-cell kinocilium. Here we show that kinocilial Pcdh15 trafficking relies on cell type-specific coupling to the generic intraflagellar transport (IFT) transport mechanism. We uncover a role for fibroblast growth factor receptor 1 (FGFR1) in loading Pcdh15 onto kinociliary transport particles in hair cells. We find that on activation, FGFR1 binds and phosphorylates Pcdh15. Moreover, we find a previously uncharacterized role for clathrin in coupling this kinocilia-specific cargo with the anterograde IFT-B complex through the adaptor, DAB2. Our results identify a modified ciliary transport pathway used for Pcdh15 transport into the cilium of the inner ear hair cell and coordinated by FGFR1 activity.

Multiple pesticide analysis in hair samples of pregnant French women: Results from the ELFE national birth cohort

BackgroundA growing body of evidence suggests that prenatal exposure to pesticides might impair fetal development. Nonetheless, knowledge about pesticide exposure of pregnant women, especially in Europe, is largely restricted to a limited panel of molecules. AimTo characterize the concentration of 140 pesticides and metabolites in hair strands from women in the ELFE French nationwide birth cohort. MethodsAmong cohort members who gave birth in northeastern and southwestern France in 2011, we selected those with a sufficient available mass of hair (n = 311). Bundles of hair 9 cm long were collected at delivery. We screened 111 pesticides and 29 metabolites, including 112 selected a priori based on their reported usage or detection in the French environment. The bundles of hair from 47 women were split into three segments to explore the intraindividual variability of the exposure. Intraclass correlation coefficients (ICCs) were computed for the chemicals with a detection frequency >70%. ResultsWe detected a median of 43 chemicals per woman (IQR 38–47). Overall, 122 chemicals (>20 chemical families) were detected at least once, including 28 chemicals detected in 70–100% of hair samples. The highest median concentrations were observed for permethrin (median: 37.9 pg/mg of hair), p-nitrophenol (13.2 pg/mg), and pentachlorophenol (10.0 pg/mg). The ICCs for the 28 chemicals studied ranged from 0.59 to 0.94. ConclusionPregnant women are exposed to multiple pesticides simultaneously from various chemical families, including chemicals suspected to be reproductive toxicants or endocrine disruptors. The ICCs suggest that the intraindividual variability of pesticide concentrations in hair is lower than its interindividual variability.

Prestin Contributes to Membrane Compartmentalization and Is Required for Normal Innervation of Outer Hair Cells.

Outer hair cells (OHC) act as amplifiers and their function is modified by medial olivocochlear (MOC) efferents. The unique OHC motor protein, prestin, provides the molecular basis for somatic electromotility, which is required for sensitivity and frequency selectivity, the hallmarks of mammalian hearing. Prestin proteins are the major component of the lateral membrane of mature OHCs, which separates apical and basal domains. To investigate the contribution of prestin to this unique arrangement, we compared the distribution of membrane proteins in OHCs of wildtype (WT) and prestin-knockout (KO) mice. In WT, the apical protein PMCA2 was exclusively localized to the hair bundles, while it was also found at the lateral membrane in KOs. Similarly, a basal protein KCNQ4 did not coalesce at the base of OHCs but was widely dispersed in mice lacking prestin. Since the expression levels of PMCA2 and KCNQ4 remained unchanged in KOs, the data indicate that prestin is required for the normal distribution of apical and basal membrane proteins in OHCs. Since OHC synapses predominate in the basal subnuclear region, we also examined the synaptic architecture in prestin-KO mice. Although neurite densities were not affected, MOC efferent terminals in prestin-KO mice were no longer constrained to the basal pole as in WT. This trend was evident as early as at postnatal day 12. Furthermore, terminals were often enlarged and frequently appeared as singlets when compared to the multiple clusters of individual terminals in WT. This abnormality in MOC synaptic morphology in prestin-KO mice is similar to defects in mice lacking MOC pathway proteins such as α9/α10 nicotinic acetylcholine receptors and BK channels, indicating a role for prestin in the proper establishment of MOC synapses. To investigate the contribution of prestin’s electromotility, we also examined OHCs from a mouse model that expresses non-functional prestin (499-prestin). We found no changes in PMCA2 localization and MOC synaptic morphology in OHCs from 499-prestin mice. Taken together, these results indicate that prestin, independent of its motile function, plays an important structural role in membrane compartmentalization, which is required for the formation of normal efferent-OHC synapses in mature OHCs.

Regenerating hair cells in vestibular sensory epithelia from humans.

Human vestibular sensory epithelia in explant culture were incubated in gentamicin to ablate hair cells. Subsequent transduction of supporting cells with ATOH1 using an Ad-2 viral vector resulted in generation of highly significant numbers of cells expressing the hair cell marker protein myosin VIIa. Cells expressing myosin VIIa were also generated after blocking the Notch signalling pathway with TAPI-1 but less efficiently. Transcriptomic analysis following ATOH1 transduction confirmed up-regulation of 335 putative hair cell marker genes, including several downstream targets of ATOH1. Morphological analysis revealed numerous cells bearing dense clusters of microvilli at the apical surfaces which showed some hair cell-like characteristics confirming a degree of conversion of supporting cells. However, no cells bore organised hair bundles and several expected hair cell markers genes were not expressed suggesting incomplete differentiation. Nevertheless, the results show a potential to induce conversion of supporting cells in the vestibular sensory tissues of humans.

Clarin-1 gene transfer rescues auditory synaptopathy in model of Usher syndrome.

Clarin-1, a tetraspan-like membrane protein defective in Usher syndrome type IIIA (USH3A), is essential for hair bundle morphogenesis in auditory hair cells. We report a new synaptic role for clarin-1 in mouse auditory hair cells elucidated by characterization of Clrn1 total (Clrn1ex4-/-) and postnatal hair cell-specific conditional (Clrn1ex4fl/fl Myo15-Cre+/-) knockout mice. Clrn1ex4-/- mice were profoundly deaf, whereas Clrn1ex4fl/fl Myo15-Cre+/- mice displayed progressive increases in hearing thresholds, with, initially, normal otoacoustic emissions and hair bundle morphology. Inner hair cell (IHC) patch-clamp recordings for the 2 mutant mice revealed defective exocytosis and a disorganization of synaptic F-actin and CaV1.3 Ca2+ channels, indicative of a synaptopathy. Postsynaptic defects were also observed, with an abnormally broad distribution of AMPA receptors associated with a loss of afferent dendrites and defective electrically evoked auditory brainstem responses. Protein-protein interaction assays revealed interactions between clarin-1 and the synaptic CaV1.3 Ca2+ channel complex via the Cavβ2 auxiliary subunit and the PDZ domain-containing protein harmonin (defective in Usher syndrome type IC). Cochlear gene therapy in vivo, through adeno-associated virus-mediated Clrn1 transfer into hair cells, prevented the synaptic defects and durably improved hearing in Clrn1ex4fl/fl Myo15-Cre+/- mice. Our results identify clarin-1 as a key organizer of IHC ribbon synapses, and suggest new treatment possibilities for USH3A patients.

Nonequilibrium limit-cycle oscillators: Fluctuations in hair bundle dynamics.

We develop a framework for the general interpretation of the stochastic dynamical system near a limit cycle. Such quasiperiodic dynamics are commonly found in a variety of nonequilibrium systems, including the spontaneous oscillations of hair cells of the inner ear. We demonstrate quite generally that in the presence of noise, the phase of the limit cycle oscillator will diffuse, while deviations in the directions locally orthogonal to that limit cycle will display the Lorentzian power spectrum of a damped oscillator. We identify two mechanisms by which these stochastic dynamics can acquire a complex frequency dependence and discuss the deformation of the mean limit cycle as a function of temperature. The theoretical ideas are applied to data obtained from spontaneously oscillating hair cells of the amphibian sacculus.

Transcriptional Dynamics of Hair-Bundle Morphogenesis Revealed with CellTrails

SUMMARYProtruding from the apical surface of inner ear sensory cells, hair bundles carry out mechanotransduction. Bundle growth involves sequential and overlapping cellular processes, which are concealed within gene expression profiles of individual cells. To dissect such processes, we developed CellTrails, a tool for uncovering, analyzing, and visualizing single-cell gene-expression dynamics. Utilizing quantitative gene-expression data for key bundle proteins from single cells of the developing chick utricle, we reconstructed de novo a bifurcating trajectory that spanned from progenitor cells to mature striolar and extrastriolar hair cells. Extraction and alignment of developmental trails and association of pseudo-time with bundle length measurements linked expression dynamics of individual genes with bundle growth stages. Differential trail analysis revealed high-resolution dynamics of transcripts that control striolar and extrastriolar bundle development, including those that encode proteins that regulate [Ca2+]i or mediate crosslinking and lengthening of actin filaments.

Molecular Composition of Vestibular Hair Bundles.

The vertebrate hair bundle, responsible for transduction of mechanical signals into receptor potentials in sensory hair cells, is an evolutionary masterpiece. Composed of actin-filled stereocilia of precisely regulated length, width, and number, the structure of the hair bundle is optimized for sensing auditory and vestibular stimuli. Recent developments in identifying the lipids and proteins constituting the hair bundle, obtained through genetics, biochemistry, and imaging, now permit a description of the consensus composition of vestibular bundles of mouse, rat, and chick.

Polymorphic Elastocapillarity: Kinetically Reconfigurable Self-Assembly of Hair Bundles by Varying the Drain Rate.

We report various patterns formed by draining liquid from hair bundles. Hair-like fibers arranged in triangular bundles self-assemble into various cross sections when immersed in liquid then removed. The combinations of their length and the kinetics, represented by the drain rate, lead to various polymorphic self-assemblies: concave hexagonal, triangular, circular, or inverted triangular patterns. The equilibrium of these shapes is predicted by elastocapillarity, the balance between the bending strain energy of the hairs and the surface energy of the liquid. Shapes with a larger strain energy, such as the inverted triangular bundles, are obtained at the higher liquid drain rates. This polymorphic self-assembly is fully reversible by rewetting and draining and can have applications in multifunctional dynamic textures.

Mitochondria-targeting nanomedicine: An effective and potent strategy against aminoglycosides-induced ototoxicity

We report a proof-of-concept for the development of mitochondria-targeting nanoparticles (NPs) loaded with geranylgeranylacetone (GGA) to protect against a wide range of gentamicin-induced ototoxicity symptoms in a zebrafish model. The polymeric NPs were functionalized with a mitochondrial-homing peptide (d‑Arg‑Dmt‑Orn‑Phe‑NH2) and exhibited greater mitochondrial uptake and lower gentamicin uptake in hair cells via mechanotransduction (MET) channels and tuned machinery in the hair bundle than the ordinary NPs did. Blockade of MET channels rapidly reversed this effect, indicating the reversible responses of hair cells to the targeting NPs were mediated by MET channels. Pretreatment of hair cells with mitochondria-targeting GGA-loaded NPs exhibited a superior acute or chronic protective efficacy against subsequent exposure to gentamicin compared with unmodified formulations. Mitochondrial delivery regulating the death pathway of hair cells appeared to cause the therapeutic failure of untargeted NPs. Thus, peptide-directed mitochondria-targeting NPs may represent a novel therapeutic strategy for mitochondrial dysfunction-linked diseases.

Directional selectivity of afferent neurons in zebrafish neuromasts is regulated by Emx2 in presynaptic hair cells.

The orientation of hair bundles on top of sensory hair cells (HCs) in neuromasts of the lateral line system allows fish to detect direction of water flow. Each neuromast shows hair bundles arranged in two opposing directions and each afferent neuron innervates only HCs of the same orientation. Previously, we showed that this opposition is established by expression of Emx2 in half of the HCs, where it mediates hair bundle reversal (<xref ref-type="bibr" rid="bib15">Jiang et al., 2017</xref>). Here, we show that Emx2 also regulates neuronal selection: afferent neurons innervate either Emx2-positive or negative HCs. In emx2 knockout and gain-of-function neuromasts, all HCs are unidirectional and the innervation patterns and physiological responses of the afferent neurons are dependent on the presence or absence of Emx2. Our results indicate that Emx2 mediates the directional selectivity of neuromasts by two distinct processes: regulating hair bundle orientation in HCs and selecting afferent neuronal targets.