Unconventional secretory pathway activation restores hair cell mechanotransduction in an USH3A model

The pathogenic variant c.144T>G (p.N48K) in the clarin1 gene (CLRN1) results in progressive loss of vision and hearing in Usher syndrome IIIA (USH3A) patients. CLRN1 is predicted to be an essential protein in hair bundles, the mechanosensory structure of hair cells critical for hearing and balance. When expressed in animal models, CLRN1 localizes to the hair bundle, whereas glycosylation-deficient CLRN1N48K aggregates in the endoplasmic reticulum, with only a fraction reaching the bundle. We hypothesized that the small amount of CLRN1N48K that reaches the hair bundle does so via an unconventional secretory pathway and that activation of this pathway could be therapeutic. Using genetic and pharmacological approaches, we find that clarin1 knockout (clrn1KO/KO) zebrafish that express the CLRN1c.144T>G pathogenic variant display progressive hair cell dysfunction, and that CLRN1N48K is trafficked to the hair bundle via the GRASP55 cargo-dependent unconventional secretory pathway (GCUSP). On expression of GRASP55 mRNA, or on exposure to the drug artemisinin (which activates GCUSP), the localization of CLRN1N48K to the hair bundles was enhanced. Artemisinin treatment also effectively restored hair cell mechanotransduction and attenuated progressive hair cell dysfunction in clrn1KO/KO larvae that express CLRN1c.144T>G, highlighting the potential of artemisinin to prevent sensory loss in CLRN1c.144T>G patients.

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Zebrafish Models for the Mechanosensory Hair Cell Dysfunction in Usher Syndrome 3 Reveal That Clarin-1 Is an Essential Hair Bundle Protein

Journal of Neuroscience ◽

10.1523/jneurosci.1096-15.2015 ◽

2015 ◽

Vol 35 (28) ◽

pp. 10188-10201 ◽

Cited By ~ 23

Author(s):

S. R. Gopal ◽

D. H.- C. Chen ◽

S.-W. Chou ◽

J. Zang ◽

S. C. F. Neuhauss ◽

...

Keyword(s):

Hair Cell ◽

Usher Syndrome ◽

Hair Bundle ◽

Cell Dysfunction ◽

Bundle Protein

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Shaker-1 mutations reveal roles for myosin VIIA in both development and function of cochlear hair cells

Development ◽

10.1242/dev.125.4.557 ◽

1998 ◽

Vol 125 (4) ◽

pp. 557-566 ◽

Cited By ~ 3

Author(s):

T. Self ◽

M. Mahony ◽

J. Fleming ◽

J. Walsh ◽

S.D. Brown ◽

...

Keyword(s):

Hair Cell ◽

Hair Cells ◽

Usher Syndrome ◽

Orthologous Gene ◽

Cell Development ◽

Cochlear Hair Cells ◽

Cochlear Hair Cell ◽

Show Evidence ◽

Myosin Viia ◽

And Function

The mouse shaker-1 locus, Myo7a, encodes myosin VIIA and mutations in the orthologous gene in humans cause Usher syndrome type 1B or non-syndromic deafness. Myo7a is expressed very early in sensory hair cell development in the inner ear. We describe the effects of three mutations on cochlear hair cell development and function. In the Myo7a816SB and Myo7a6J mutants, stereocilia grow and form rows of graded heights as normal, but the bundles become progressively more disorganised. Most of these mutants show no gross electrophysiological responses, but some did show evidence of hair cell depolarisation despite the disorganisation of their bundles. In contrast, the original shaker-1 mutants, Myo7ash1, had normal early development of stereocilia bundles, but still showed abnormal cochlear responses. These findings suggest that myosin VIIA is required for normal stereocilia bundle organisation and has a role in the function of cochlear hair cells.

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Comparative transduction mechanisms of hair cells in the bullfrog utriculus. I. Responses to intracellular current

Journal of Neurophysiology ◽

10.1152/jn.1994.71.2.666 ◽

1994 ◽

Vol 71 (2) ◽

pp. 666-684 ◽

Cited By ~ 20

Author(s):

R. A. Baird

Keyword(s):

B Cells ◽

Hair Cell ◽

Hair Cells ◽

Hair Bundle ◽

C Cells ◽

Type B ◽

Lateral Border ◽

Utricular Macula ◽

Type C ◽

Current Steps

1. Hair cells in whole-mount in vitro preparations of the utricular macula of the bullfrog (Rana catesbeiana) were selected according to their macular location and hair bundle morphology. The voltage responses of selected hair cells to intracellular current steps and sinusoids in the frequency range of 0.5-200 Hz were studied with conventional intracellular recordings. 2. The utricular macula is divided into medial and lateral parts by the striola, a 75- to 100-microns zone that runs for nearly the entire length of the sensory macula near its lateral border. The striola is distinguished from flanking extrastriolar regions by the elevated height of its apical surface and the wider spacing of its hair cells. A line dividing hair cells of opposing polarities, located near the lateral border of the striola, separates it into medial and lateral parts. On average, the striola consists of five to seven medial and two to three lateral rows of hair cells. 3. Utricular hair cells were classified into four types on the basis of hair bundle morphology. Type B cells, the predominant hair cell type in the utricular macula, are small cells with short sterocilia and kinocilia 2-6 times as long as their longest stereocilia. These hair cells were found throughout the extrastriola and, more rarely, in the striolar region. Three other hair cell types were restricted to the striolar region. Type C cells, found primarily in the outer striolar rows, resemble enlarged versions of Type B hair cells. Type F cells have kinocilia approximately equal in length to their longest stereocilia and are restricted to the middle striolar rows. Type E cells, found only in the innermost striolar rows, have short kinocilia with prominent kinociliary bulbs. 4. The resting potential of 99 hair cells was -58.0 +/- 7.6 (SD) mV and did not vary significantly for hair cells in differing macular locations or with differing hair bundle morphology. The RN of hair cells, measured from the voltage response to current steps, varied from 200 to > 2,000 M omega and was not well correlated with cell size. On average, Type B cells had the highest RN, followed by Type F, Type E, and Type C cells. When normalized to their surface area, the membrane resistance of hair cells ranged from < 1,000 to > 10,000 k omega.cm2. The input capacitance of hair cells ranged from < 3 to > 15 pA, corresponding on average to a membrane capacitance of 0.8 +/- 0.2 pA/cm2.(ABSTRACT TRUNCATED AT 400 WORDS)

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The development of cooperative channels explains the maturation of hair cell’s mechanotransduction

10.1101/654764 ◽

2019 ◽

Author(s):

Francesco Gianoli ◽

Thomas Risler ◽

Andrei S. Kozlov

Keyword(s):

Ion Channels ◽

Hair Cell ◽

Inner Ear ◽

Hair Cells ◽

Hair Bundle ◽

Mechanical Stimuli ◽

Biophysical Properties ◽

Tip Link ◽

Fast Adaptation ◽

Kinetics Of

ABSTRACTHearing relies on the conversion of mechanical stimuli into electrical signals. In vertebrates, this process of mechano-electrical transduction (MET) is performed by specialized receptors of the inner ear, the hair cells. Each hair cell is crowned by a hair bundle, a cluster of microvilli that pivot in response to sound vibrations, causing the opening and closing of mechanosensitive ion channels. Mechanical forces are projected onto the channels by molecular springs called tip links. Each tip link is thought to connect to a small number of MET channels that gate cooperatively and operate as a single transduction unit. Pushing the hair bundle in the excitatory direction opens the channels, after which they rapidly reclose in a process called fast adaptation. It has been experimentally observed that the hair cell’s biophysical properties mature gradually during postnatal development: the maximal transduction current increases, sensitivity sharpens, transduction occurs at smaller hair-bundle displacements, and adaptation becomes faster. Similar observations have been reported during tip-link regeneration after acoustic damage. Moreover, when measured at intermediate developmental stages, the kinetics of fast adaptation varies in a given cell depending on the magnitude of the imposed displacement. The mechanisms underlying these seemingly disparate observations have so far remained elusive. Here, we show that these phenomena can all be explained by the progressive addition of MET channels of constant properties, which populate the hair bundle first as isolated entities, then progressively as clusters of more sensitive, cooperative MET channels. As the proposed mechanism relies on the difference in biophysical properties between isolated and clustered channels, this work highlights the importance of cooperative interactions between mechanosensitive ion channels for hearing.SIGNIFICANCEHair cells are the sensory receptors of the inner ear that convert mechanical stimuli into electrical signals transmitted to the brain. Sensitivity to mechanical stimuli and the kinetics of mechanotransduction currents change during hair-cell development. The same trend, albeit on a shorter timescale, is also observed during hair-cell recovery from acoustic trauma. Furthermore, the current kinetics in a given hair cell depends on the stimulus magnitude, and the degree of that dependence varies with development. These phenomena have so far remained unexplained. Here, we show that they can all be reproduced using a single unifying mechanism: the progressive formation of channel pairs, in which individual channels interact through the lipid bilayer and gate cooperatively.

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Multiple PDZ domain protein maintains patterning of the apical cytoskeleton in sensory hair cells

Development ◽

10.1242/dev.199549 ◽

2021 ◽

Author(s):

Amandine Jarysta ◽

Basile Tarchini

Keyword(s):

Hair Cell ◽

Hair Cells ◽

Critical Role ◽

Pdz Domain ◽

Hair Bundle ◽

Structural Defects ◽

Sensory Hair ◽

High Frequencies ◽

Sensory Hair Cells ◽

Sound Transduction

Sound transduction occurs in the hair bundle, the apical compartment of sensory hair cells in the inner ear. The hair bundle is formed of actin-based stereocilia aligned in rows of graded heights. It was previously shown that the GNAI-GPSM2 complex is part of a developmental blueprint that defines the polarized organization of the apical cytoskeleton in hair cells, including stereocilia distribution and elongation. Here we report a novel and critical role for Multiple PDZ domain (MPDZ) protein during apical hair cell morphogenesis. We show that MPDZ is enriched at the hair cell apical membrane along with MAGUK p55 subfamily member 5 (MPP5/PALS1) and the Crumbs protein CRB3. MPDZ is required there to maintain the proper segregation of apical blueprints proteins, including GNAI-GPSM2. Loss of the blueprint coincides with misaligned stereocilia placement in Mpdz mutant hair cells, and results in permanently misshapen hair bundles. Graded molecular and structural defects along the cochlea can explain the profile of hearing loss in Mpdz mutants, where deficits are most severe at high frequencies.

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Dynamics of mechanically coupled hair-cell bundles of the inner ear

10.1101/2020.08.20.259507 ◽

2020 ◽

Author(s):

Y. Roongthumskul ◽

J. Faber ◽

D. Bozovic

Keyword(s):

Inner Ear ◽

Auditory System ◽

Hair Cells ◽

Coupled System ◽

Acoustic Energy ◽

Hair Bundle ◽

Free Standing ◽

Physiological Level ◽

Mechanical Coupling ◽

Hair Bundles

ABSTRACTThe high sensitivity and effective frequency discrimination of sound detection performed by the auditory system rely on the dynamics of a system of hair cells. In the inner ear, these acoustic receptors are primarily attached to an overlying structure which provides mechanical coupling between the hair bundles. While the dynamics of individual hair bundles have been extensively investigated, the influence of mechanical coupling on the motility of the system of bundles remains underdetermined. We developed a technique of mechanically coupling two active hair bundles, enabling us to probe the dynamics of the coupled system experimentally. We demonstrated that the coupling could enhance the coherence of hair bundles’ spontaneous oscillation as well as their phase-locked response to sinusoidal stimuli, at the calcium concentration in the surrounding fluid near the physiological level. The empirical data were consistent with numerical results from a model of two coupled nonisochronous oscillators, each displaying a supercritical Hopf bifurcation. The model revealed that weak coupling can poise the system of unstable oscillators closer to the bifurcation by a shift in the critical point. In addition, the dynamics of strongly coupled oscillators far from criticality suggested that individual hair bundles may be regarded as nonisochronous oscillators. An optimal degree of nonisochronicity was required for the observed tuning behavior in the coherence of autonomous motion of the coupled system.STATEMENT OF SIGNIFICANCEHair cells of the inner ear transduce acoustic energy into electrical signals via a deflection of hair bundles. Unlike a passive mechanical antenna, a free-standing hair bundle behaves as an active oscillator that can sustain autonomous oscillations, as well as amplify a low-level stimulus. Hair bundles under physiological conditions are elastically coupled to each other via an extracellular matrix. Therefore, the dynamics of coupled nonlinear oscillators underlie the performance of the peripheral auditory system. Despite extensive theoretical investigations, there are limited experimental evidence that support the significance of coupling on hair bundle motility. We develop a technique to mechanically couple hair bundles and demonstrate the benefits of coupling on hair bundle spontaneous motility.

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Par3 is essential for the establishment of planar cell polarity of inner ear hair cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1816333116 ◽

2019 ◽

Vol 116 (11) ◽

pp. 4999-5008 ◽

Cited By ~ 11

Author(s):

Andre Landin Malt ◽

Zachary Dailey ◽

Julia Holbrook-Rasmussen ◽

Yuqiong Zheng ◽

Arielle Hogan ◽

...

Keyword(s):

Hair Cell ◽

Inner Ear ◽

Cell Polarity ◽

Hair Cells ◽

Planar Cell Polarity ◽

Tissue Level ◽

Hair Bundle ◽

Nucleotide Exchange ◽

Genetic Mosaic ◽

Pcp Signaling

In the inner ear sensory epithelia, stereociliary hair bundles atop sensory hair cells are mechanosensory apparatus with planar polarized structure and orientation. This is established during development by the concerted action of tissue-level, intercellular planar cell polarity (PCP) signaling and a hair cell-intrinsic, microtubule-mediated machinery. However, how various polarity signals are integrated during hair bundle morphogenesis is poorly understood. Here, we show that the conserved cell polarity protein Par3 is essential for planar polarization of hair cells. Par3 deletion in the inner ear disrupted cochlear outgrowth, hair bundle orientation, kinocilium positioning, and basal body planar polarity, accompanied by defects in the organization and cortical attachment of hair cell microtubules. Genetic mosaic analysis revealed that Par3 functions both cell-autonomously and cell-nonautonomously to regulate kinocilium positioning and hair bundle orientation. At the tissue level, intercellular PCP signaling regulates the asymmetric localization of Par3, which in turn maintains the asymmetric localization of the core PCP protein Vangl2. Mechanistically, Par3 interacts with and regulates the localization of Tiam1 and Trio, which are guanine nucleotide exchange factors (GEFs) for Rac, thereby stimulating Rac-Pak signaling. Finally, constitutively active Rac1 rescued the PCP defects in Par3-deficient cochleae. Thus, a Par3–GEF–Rac axis mediates both tissue-level and hair cell-intrinsic PCP signaling.

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EMX2-GPR156-Gαi reverses hair cell orientation in mechanosensory epithelia

Nature Communications ◽

10.1038/s41467-021-22997-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Katie S. Kindt ◽

Anil Akturk ◽

Amandine Jarysta ◽

Matthew Day ◽

Alisha Beirl ◽

...

Keyword(s):

Hair Cell ◽

Hair Cells ◽

Mirror Image ◽

Hair Bundle ◽

Otolith Organs ◽

Cell Orientation ◽

Stimulus Detection ◽

Cell Reversal ◽

Image Organization ◽

Orphan Gpcr

AbstractHair cells detect sound, head position or water movements when their mechanosensory hair bundle is deflected. Each hair bundle has an asymmetric architecture that restricts stimulus detection to a single axis. Coordinated hair cell orientations within sensory epithelia further tune stimulus detection at the organ level. Here, we identify GPR156, an orphan GPCR of unknown function, as a critical regulator of hair cell orientation. We demonstrate that the transcription factor EMX2 polarizes GPR156 distribution, enabling it to signal through Gαi and trigger a 180° reversal in hair cell orientation. GPR156-Gαi mediated reversal is essential to establish hair cells with mirror-image orientations in mouse otolith organs in the vestibular system and in zebrafish lateral line. Remarkably, GPR156-Gαi also instructs hair cell reversal in the auditory epithelium, despite a lack of mirror-image organization. Overall, our work demonstrates that conserved GPR156-Gαi signaling is integral to the framework that builds directional responses into mechanosensory epithelia.

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Dimensions of a Living Cochlear Hair Bundle

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.742529 ◽

2021 ◽

Vol 9 ◽

Author(s):

Katharine K. Miller ◽

Patrick Atkinson ◽

Kyssia Ruth Mendoza ◽

Dáibhid Ó Maoiléidigh ◽

Nicolas Grillet

Keyword(s):

Hair Cells ◽

Fluid Flows ◽

Cell Types ◽

Fluorescent Imaging ◽

Hair Bundle ◽

Mechanical Stimuli ◽

Accuracy And Precision ◽

Limited Degree ◽

Fluid Coupling ◽

Hair Bundles

The hair bundle is the mechanosensory organelle of hair cells that detects mechanical stimuli caused by sounds, head motions, and fluid flows. Each hair bundle is an assembly of cellular-protrusions called stereocilia, which differ in height to form a staircase. Stereocilia have different heights, widths, and separations in different species, sensory organs, positions within an organ, hair-cell types, and even within a single hair bundle. The dimensions of the stereociliary assembly dictate how the hair bundle responds to stimuli. These hair-bundle properties have been measured previously only to a limited degree. In particular, mammalian data are either incomplete, lack control for age or position within an organ, or have artifacts owing to fixation or dehydration. Here, we provide a complete set of measurements for postnatal day (P) 11 C57BL/6J mouse apical inner hair cells (IHCs) obtained from living tissue, tissue mildly-fixed for fluorescent imaging, or tissue strongly fixed and dehydrated for scanning electronic microscopy (SEM). We found that hair bundles mildly-fixed for fluorescence had the same dimensions as living hair bundles, whereas SEM-prepared hair bundles shrank uniformly in stereociliary heights, widths, and separations. By determining the shrinkage factors, we imputed live dimensions from SEM that were too small to observe optically. Accordingly, we created the first complete blueprint of a living IHC hair bundle. We show that SEM-prepared measurements strongly affect calculations of a bundle’s mechanical properties – overestimating stereociliary deflection stiffness and underestimating the fluid coupling between stereocilia. The methods of measurement, the data, and the consequences we describe illustrate the high levels of accuracy and precision required to understand hair-bundle mechanotransduction.

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Multiple PDZ Domain Protein Maintains Patterning of the Apical Cytoskeleton in Sensory Hair Cells

10.1101/2021.02.20.432099 ◽

2021 ◽

Author(s):

Amandine Jarysta ◽

Basile Tarchini

Keyword(s):

Hair Cell ◽

Hair Cells ◽

Critical Role ◽

Pdz Domain ◽

Hair Bundle ◽

Structural Defects ◽

Sensory Hair ◽

High Frequencies ◽

Sensory Hair Cells ◽

Sound Transduction

SUMMARYSound transduction occurs in the hair bundle, the apical compartment of sensory hair cells in the inner ear. The hair bundle is formed of stereocilia aligned in rows of graded heights. It was previously shown that the GNAI-GPSM2 complex is part of a developmental blueprint that defines the polarized organization of the apical cytoskeleton in hair cells, including stereocilia distribution and elongation. Here we report a novel and critical role for Multiple PDZ domain (MPDZ) protein during apical hair cell morphogenesis. We show that MPDZ is enriched at the hair cell apical membrane, and required there to maintain the proper segregation of apical blueprints proteins, including GNAI-GPSM2. Loss of the blueprint coincides with misaligned stereocilia in Mpdz mutants, and results in permanently misshapen hair bundles. Graded molecular and structural defects along the cochlea can explain the profile of hearing loss in Mpdz mutants, where deficits are most severe at high frequencies.

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