Harmonin-b, an actin-binding scaffold protein, is involved in the adaptation of mechanoelectrical transduction by sensory hair cells

We assessed the involvement of harmonin-b, a submembranous protein containing PDZ domains, in the mechanoelectrical transduction machinery of inner ear hair cells. Harmonin-b is located in the region of the upper insertion point of the tip link that joins adjacent stereocilia from different rows and that is believed to gate transducer channel(s) located in the region of the tip link's lower insertion point. In Ush1cdfcr-2J/dfcr-2J mutant mice defective for harmonin-b, step deflections of the hair bundle evoked transduction currents with altered speed and extent of adaptation. In utricular hair cells, hair bundle morphology and maximal transduction currents were similar to those observed in wild-type mice, but adaptation was faster and more complete. Cochlear outer hair cells displayed reduced maximal transduction currents, which may be the consequence of moderate structural anomalies of their hair bundles. Their adaptation was slower and displayed a variable extent. The latter was positively correlated with the magnitude of the maximal transduction current, but the cells that showed the largest currents could be either hyperadaptive or hypoadaptive. To interpret our observations, we used a theoretical description of mechanoelectrical transduction based on the gating spring theory and a motor model of adaptation. Simulations could account for the characteristics of transduction currents in wild-type and mutant hair cells, both vestibular and cochlear. They led us to conclude that harmonin-b operates as an intracellular link that limits adaptation and engages adaptation motors, a dual role consistent with the scaffolding property of the protein and its binding to both actin filaments and the tip link component cadherin-23

Machinerie de transduction mécano-électrique de l'oreille interne (caractérisation fonctionnelle et mécanismes moléculaires sous-jacents)

J ai étudié le rôle de sans, protéine responsable de la forme USH1G du syndrome d Usher, dans la transduction mécano-électrique des cellules ciliées de l oreille interne, en utilisant la technique du patch clamp, couplé à la stimulation mécanique de ces cellules. Une déflection de la touffe ciliaire augmente la tension exercée sur le tip-link, lien qui contrôle la probabilité d ouverture du canal de transduction. Chez les souris Ush1g-/-, la structure anormale de la touffe ciliaire s accompagne d une diminution prononcée des courants de transduction. Pour s affranchir de ces défauts morphologiques, nous avons produit une souris Ush1gfl/flMyo15-cre+/-, chez laquelle Ush1g est inactivé après la naissance. Ces souris, ont une diminution de l'amplitude du courant de transduction, accompagnée de la perte des tip-links. En outre, certains des stéréocils, qui composent la touffe ciliaire, régressent jusqu à disparaitre. Nous avons pu conclure que sans appartient au complexe de transduction, joue un rôle essentiel dans le maintien des tip-links, et que la tension des tip-links est nécessaire au maintien de la longueur des stéréocils des petite et moyenne rangéesPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Effects of a skin-massaging device on the <i>ex-vivo</i> expression of human dermis proteins and <i>in-vivo</i> facial wrinkles

<div><p>Mechanical and geometrical cues influence cell behaviour. At the tissue level, almost all organs exhibit immediate mechanical responsiveness, in particular by increasing their stiffness in direct proportion to an applied mechanical stress. It was recently shown in cultured-cell models, in particular with fibroblasts, that the frequency of the applied stress is a fundamental stimulating parameter. However, the influence of the stimulus frequency at the tissue level has remained elusive. Using a device to deliver an oscillating torque that generates cyclic strain at different frequencies, we studied the effect(s) of mild skin massage in an <i>ex vivo</i> model and <i>in vivo</i>. Skin explants were maintained <i>ex vivo</i> for 10 days and massaged twice daily for one minute at various frequencies within the range of 65–85 Hz. Biopsies were analysed at D0, D5 and D10 and processed for immuno-histological staining specific to various dermal proteins. As compared to untreated skin explants, the massaging procedure clearly led to higher rates of expression, in particular for decorin, fibrillin, tropoelastin, and procollagen-1. The mechanical stimulus thus evoked an anti-aging response. Strikingly, the expression was found to depend on the stimulus frequency with maximum expression at 75Hz. We then tested whether this mechanical stimulus had an anti-aging effect <i>in vivo</i>. Twenty Caucasian women (aged 65-75y) applied a commercial anti-aging cream to the face and neck, followed by daily treatments using the anti-aging massage device for 8 weeks. A control group of twenty-two women, with similar ages to the first group, applied the cream alone. At W0, W4 and W8, a blinded evaluator assessed the global facial wrinkles, skin texture, lip area, cheek wrinkles, neck sagging and neck texture using a clinical grading scale. We found that combining the massaging device with a skin anti-aging formulation amplified the beneficial effects of the cream.</p></div

Determination of frequency and amplitude of the massaging device <i>in vivo</i>.

<p>The frequency and amplitude was verified <i>in vivo</i> in the forearm with ultrafast ultrasound imaging. (A) Spatiotemporal spreading of vibrations generated by the device at 65 Hz in the forearm of healthy volunteer of 26yo. The frequency and the amplitude of the displacement imposed to the skin were measured at a point 25 mm far from the source and 500μm depth on the tissue (the exact position is marked with an arrow). (B) The amplitude of the vibration measured the point marked in pink in <i>A</i> is about 150μm peak to peak. (C) The spectrum of the displacement field (averaged calculated in the full dermis and expressed in Arbitrary Units) shows that the fundamental frequency is 65 ± 3 Hz. (D) The spectrum of the displacement field (averaged calculated in the full dermis and expressed in Arbitrary Units) shows that the fundamental frequency is 85 ± 3 Hz.</p

Immuno-labeling of DEJ and Dermal proteins for 75Hz treatment.

<p>(A) Immuno-labeling of some dermal markers (green labeling); (B)Box Plot representation of the fluorescence intensity (arbitrary scale) of the measured markers. Data shown are collected from one 68 year old donor, after 5 days of massaging for all markers, with the exception of type VII collagen and procollagen 1, which were sampled after 10 days of treatment from the same donor. A second 50 year old donor was analyzed with similar results (data not shown). The stars indicate the statistical significance of the labeling quantification for each condition compared to untreated/control skin (***: p<0.001, **: p<0.01 *: p<0.05).</p

Global wrinkle changes during study.

<p>Example of two volunteers in Group 2 (cream plus sonic skin-massaging device) before (baseline), after 4 weeks of treatment and after 8 weeks of treatment. Standardized photographs illustrate an improvement of global facial wrinkles. Arrows point out specific areas of interest.</p

Summary of the various antibodies/protein label used in the ex vivo histological study.

<p>Summary of the various antibodies/protein label used in the ex vivo histological study.</p

Treatment system.

<p>(A) A technical drawing of the massage head, showing the distance between the contact points of 2,5cm. (B): The massage head with 3 contact points used in <i>in vivo</i> study. (C) The massage head used in the ex vivo experiments, with only two massage points spaced 2,5cm apart. (D) The configuration set up for the ex vivo experiments: The Smart PROfile handle is connected to a micrometer staging system to control the position of the device on the skin. The pressure applied was measured with a scale and standardized at 80g. The entire set up was mounted on an anti-vibration table.</p