Mutations in cadherin 23 affect tip links in zebrafish sensory hair cells

Hair cells have highly organized bundles of apical projections, or stereocilia, that are deflected by sound and movement. Displacement of stereocilia stretches linkages at the tips of stereocilia that are thought to gate mechanosensory channels. To identify the molecular machinery that mediates mechanotransduction in hair cells, zebrafish mutants were identified with defects in balance and hearing. In sputnik mutants, stereociliary bundles are splayed to various degrees, with individuals displaying reduced or absent mechanotransduction. Here we show that the defects in sputnik mutants are caused by mutations in cadherin 23 (cdh23). Mutations in Cdh23 also cause deafness and vestibular defects in mice and humans, and the protein is present in hair bundles. We show that zebrafish Cdh23 protein is concentrated near the tips of hair bundles, and that tip links are absent in homozygous sputnik(tc317e) larvae. Moreover, tip links are absent in larvae carrying weak alleles of cdh23 that affect mechanotransduction but not hair bundle integrity. We conclude that Cdh23 is an essential tip link component required for hair-cell mechanotransduction.     

Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia

Hair cells of the inner ear are not normally replaced during an animal's life, and must continually renew components of their various organelles. Among these are the stereocilia, each with a core of several hundred actin filaments that arise from their apical surfaces and that bear the mechanotransduction apparatus at their tips. Actin turnover in stereocilia has previously been studied by transfecting neonatal rat hair cells in culture with a β-actin-GFP fusion, and evidence was found that actin is replaced, from the top down, in 2-3 days. Overexpression of the actin-binding protein espin causes elongation of stereocilia within 12-24 hours, also suggesting rapid regulation of stereocilia lengths. Similarly, the mechanosensory 'tip links' are replaced in 5-10 hours after cleavage in chicken and mammalian hair cells. In contrast, turnover in chick stereocilia in vivo is much slower. It might be that only certain components of stereocilia turn over quickly, that rapid turnover occurs only in neonatal animals, only in culture, or only in response to a challenge like breakage or actin overexpression. Here we quantify protein turnover by feeding animals with a (15)N-labelled precursor amino acid and using multi-isotope imaging mass spectrometry to measure appearance of new protein. Surprisingly, in adult frogs and mice and in neonatal mice, in vivo and in vitro, the stereocilia were remarkably stable, incorporating newly synthesized protein at <10% per day. Only stereocilia tips had rapid turnover and no treadmilling was observed. Other methods confirmed this: in hair cells expressing β-actin-GFP we bleached fiducial lines across hair bundles, but they did not move in 6 days. When we stopped expression of β- or γ-actin with tamoxifen-inducible recombination, neither actin isoform left the stereocilia, except at the tips. Thus, rapid turnover in stereocilia occurs only at the tips and not by a treadmilling process.     

Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells

Hair cells of the inner ear are mechanosensors that transduce mechanical forces arising from sound waves and head movement into electrochemical signals to provide our sense of hearing and balance. Each hair cell contains at the apical surface a bundle of stereocilia. Mechanoelectrical transduction takes place close to the tips of stereocilia in proximity to extracellular tip-link filaments that connect the stereocilia and are thought to gate the mechanoelectrical transduction channel. Recent reports on the composition, properties and function of tip links are conflicting. Here we demonstrate that two cadherins that are linked to inherited forms of deafness in humans interact to form tip links. Immunohistochemical studies using rodent hair cells show that cadherin 23 (CDH23) and protocadherin 15 (PCDH15) localize to the upper and lower part of tip links, respectively. The amino termini of the two cadherins co-localize on tip-link filaments. Biochemical experiments show that CDH23 homodimers interact in trans with PCDH15 homodimers to form a filament with structural similarity to tip links. Ions that affect tip-link integrity and a mutation in PCDH15 that causes a recessive form of deafness disrupt interactions between CDH23 and PCDH15. Our studies define the molecular composition of tip links and provide a conceptual base for exploring the mechanisms of sensory impairment associated with mutations in CDH23 and PCDH15.     

How the ear's works work

The senses of hearing and equilibrium depend on sensory receptors called hair cells which can detect motions of atomic dimensions and respond more than 100,000 times a second. Biophysical studies suggest that mechanical forces control the opening and closing of transduction channels by acting through elastic components in each hair cell's mechanoreceptive hair bundle. Other ion channels, as well as the mechanical and hydrodynamic properties of hair bundles, tune individual hair cells to particular frequencies of stimulation.     

Cadherin 23 is a component of the tip link in hair-cell stereocilia

Mechanoelectrical transduction, the conversion of mechanical force into electrochemical signals, underlies a range of sensory phenomena, including touch, hearing and balance. Hair cells of the vertebrate inner ear are specialized mechanosensors that transduce mechanical forces arising from sound waves and head movement to provide our senses of hearing and balance; however, the mechanotransduction channel of hair cells and the molecules that regulate channel activity have remained elusive. One molecule that might participate in mechanoelectrical transduction is cadherin 23 (CDH23), as mutations in its gene cause deafness and age-related hearing loss. Furthermore, CDH23 is large enough to be the tip link, the extracellular filament proposed to gate the mechanotransduction channel. Here we show that antibodies against CDH23 label the tip link, and that CDH23 has biochemical properties similar to those of the tip link. Moreover, CDH23 forms a complex with myosin-1c, the only known component of the mechanotransduction apparatus, suggesting that CDH23 and myosin-1c cooperate to regulate the activity of mechanically gated ion channels in hair cells.     

Force generation by mammalian hair bundles supports a role in cochlear amplification

It is generally accepted that the acute sensitivity and frequency discrimination of mammalian hearing requires active mechanical amplification of the sound stimulus within the cochlea. The prevailing hypothesis is that this amplification stems from somatic electromotility of the outer hair cells attributable to the motor protein prestin. Thus outer hair cells contract and elongate in synchrony with the sound-evoked receptor potential. But problems arise with this mechanism at high frequencies, where the periodic component of the receptor potential will be attenuated by the membrane time constant. On the basis of work in non-mammalian vertebrates, force generation by the hair bundles has been proposed as an alternative means of boosting the mechanical stimulus. Here we show that hair bundles of mammalian outer hair cells can also produce force on a submillisecond timescale linked to adaptation of the mechanotransducer channels. Because the bundle motor may ultimately be limited by the deactivation rate of the channels, it could theoretically operate at high frequencies. Our results show the existence of another force generator in outer hair cells that may participate in cochlear amplification.     

Adhesion: elastocapillary coalescence in wet hair

We investigated why wet hair clumps into bundles by dunking a model brush of parallel elastic lamellae into a perfectly wetting liquid. As the brush is withdrawn, pairs of bundles aggregate successively, forming complex hierarchical patterns that depend on a balance between capillary forces and the elasticity of the lamellae. This capillary-driven self-assembly of flexible structures, which occurs in the tarsi of insects and in biomimetic adhesives but which can also damage micro-electromechanical structures or carbon nanotube 'carpets', represents a new type of coalescence process.     

TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells

Mechanical deflection of the sensory hair bundles of receptor cells in the inner ear causes ion channels located at the tips of the bundle to open, thereby initiating the perception of sound. Although some protein constituents of the transduction apparatus are known, the mechanically gated transduction channels have not been identified in higher vertebrates. Here, we investigate TRP (transient receptor potential) ion channels as candidates and find one, TRPA1 (also known as ANKTM1), that meets criteria for the transduction channel. The appearance of TRPA1 messenger RNA expression in hair cell epithelia coincides developmentally with the onset of mechanosensitivity. Antibodies to TRPA1 label hair bundles, especially at their tips, and tip labelling disappears when the transduction apparatus is chemically disrupted. Inhibition of TRPA1 protein expression in zebrafish and mouse inner ears inhibits receptor cell function, as assessed with electrical recording and with accumulation of a channel-permeant fluorescent dye. TRPA1 is probably a component of the transduction channel itself.     

气泡粘性对上升运动特性影响的FTM模拟

为了研究气泡粘性对其上升过程的速度、形态以及阻力系数的影响,基于FTM(Front- Tracking Method)模拟了气泡在粘性流体中的运动,通过改变气泡粘性来改变气液粘度比,研究了气泡内的速度分布以及气泡周围粘性剪切应力的分布和变化特点。研究表明：气液粘度比较小时,流场中的粘性耗散很强,气泡的粘度主要影响其平衡速度和形态,气液粘度比较大时,粘性耗散减弱,其平衡速度和形态基本无变化,气泡粘性主要影响气泡内的速度分布;气泡上升过程中的阻力系数随粘度比增大而减小,粘性剪切应力则呈现相反的变化趋势。气泡上升过程中,周围的粘性剪切应力关于中心轴线对称分布,出现最大粘性切应力的区域由气泡两侧逐渐向气泡底部边缘过渡;气泡尾部边缘的形变引起气泡尾部的表面张力不平衡,使得气泡的形态发生改变。     

Rapid renewal of auditory hair bundles

Stereocilia, also known as hair bundles, are mechanosensitive organelles of the sensory hair cells of the inner ear that can detect displacements on a nanometre scale and are supported by a rigid, dense core of actin filaments. Here we show that these actin-filament arrays are continuously remodelled by the addition of actin monomers to the stereocilium tips, and that the entire core of the stereocilium is renewed every 48 hours. This unexpected dynamic feature of stereocilia will help our understanding of how auditory sensory function develops and is maintained.     

Mechanoelectrical transduction of adult outer hair cells studied in a gerbil hemicochlea

Sensory receptor cells of the mammalian cochlea are morphologically and functionally dichotomized. Inner hair cells transmit auditory information to the brain, whereas outer hair cells (OHC) amplify the mechanical signal, which is then transduced by inner hair cells. Amplification by OHCs is probably mediated by their somatic motility in a mechanical feedback process. OHC motility in vivo is thought to be driven by the cell's receptor potential. The first steps towards the generation of the receptor potential are the deflection of the stereociliary bundle, and the subsequent flow of transducer current through the mechanosensitive transducer channels located at their tips. Quantitative relations between transducer currents and basilar membrane displacements are lacking, as well as their variation along the cochlear length. To address this, we simultaneously recorded OHC transducer currents (or receptor potentials) and basilar membrane motion in an excised and bisected cochlea, the hemicochlea. This preparation permits recordings from adult OHCs at various cochlear locations while the basilar membrane is mechanically stimulated. Furthermore, the stereocilia are deflected by the same means of stimulation as in vivo. Here we show that asymmetrical transducer currents and receptor potentials are significantly larger than previously thought, they possess a highly restricted dynamic range and strongly depend on cochlear location.     

Stereocilin-deficient mice reveal the origin of cochlear waveform distortions

Although the cochlea is an amplifier and a remarkably sensitive and finely tuned detector of sounds, it also produces conspicuous mechanical and electrical waveform distortions. These distortions reflect nonlinear mechanical interactions within the cochlea. By allowing one tone to suppress another (masking effect), they contribute to speech intelligibility. Tones can also combine to produce sounds with frequencies not present in the acoustic stimulus. These sounds compose the otoacoustic emissions that are extensively used to screen hearing in newborns. Because both cochlear amplification and distortion originate from the outer hair cells-one of the two types of sensory receptor cells-it has been speculated that they stem from a common mechanism. Here we show that the nonlinearity underlying cochlear waveform distortions relies on the presence of stereocilin, a protein defective in a recessive form of human deafness. Stereocilin was detected in association with horizontal top connectors, lateral links that join adjacent stereocilia within the outer hair cell's hair bundle. These links were absent in stereocilin-null mutant mice, which became progressively deaf. At the onset of hearing, however, their cochlear sensitivity and frequency tuning were almost normal, although masking was much reduced and both acoustic and electrical waveform distortions were completely lacking. From this unique functional situation, we conclude that the main source of cochlear waveform distortions is a deflection-dependent hair bundle stiffness resulting from constraints imposed by the horizontal top connectors, and not from the intrinsic nonlinear behaviour of the mechanoelectrical transducer channel.     

The study on the relationship between breakup modes and gas-liquid interfaces

Based on the linear instability analysis, the study on the relationship between breakup modes and gas-liquid Interfaces of a viscous annular liquid jet moving in two swirling gas streams has been carried out. From the numerical results of the dispersion equation, the relevancy of the breakup mode between an annular liquid jet and two liquid jets of limiting cases, namely the cylindrical liquid jet and hollow gas jet, as well as the effects of injecting factors on the instability of an annular liquid jet, is studied in detail. Considering the effects of inner and outer interface radii on the instability of the jet, it is proved that the pars-sinuous mode mainly relates to the inner interface, whereas the pars-varicose mode mainly relates to the outer interface. The results also indicate that all the forces produced by liquid jet have similar impacts on either the instability of pars-sinuous mode or pars-varicose mode due to the fact that they can affect both inner and outer gas-liquid interfaces. On the other hand, all the forces exerting only on the inner interface have more powerful effects on the instability of pars-sinuous mode, and all the forces exerting only on the outer interface have more powerful effects on the instability of pars-varicose mode. That is to say, the effects of forces are weakened greatly when penetrating the liquid jet.     

Fluid-Structure Interaction During Large Amplitude Sloshing and TLD Vibration Control

The arbitrary Lagrange-Eulerian (ALE) finite element method (FEM) was successfully used to analyze fluid-structure interaction with a free surface. The fluid was regarded as a convection dominated incompressible viscous with the viscous and the slip boundary conditions. Generalized variational principles were established for the problem with large amplitude sloshing due to the free fluid surface. The Newmark-β integration method with a predictor-corrector scheme was used to solve the nonlinear dynamic response of the coupled ALE-FEM equations. Numerical examples were given to analyze the effects of a tuned liquid damper (TLD) setting on the structure. The horizontal nonlinear displacement responses in time domain at the top of the structure and the fluid elevation histories along the wall were computed and compared with predictions of a simplified mass-spring system.     

泡状流相分布及湍流结构的欧拉-拉格朗日双向耦合数值研究

使用欧拉-拉格朗日双向耦合数值模拟方法对泡状流的相分布及气泡对液相湍流产生的影响做了详细的调查.液相的速度场采用直接数值模拟方法求解,气泡的运动轨迹由牛顿运动方程计算.相间的耦合是把相间作用力作为液相动量方程的源相来实现的,所考虑的相间作用力有阻力、剪切升力、惯性力等.研究表明:阻力、浮力、剪切升力和虚拟质量力对气泡的运动具有重要影响,大量的气泡在剪切升力的作用下聚集在壁面附近;与单液体相相比,气泡的加入使液相的平均速度、湍流强度和雷诺应力均有一定幅度的降低,而且气泡的加入修改了阻力系数公式.     

欧活血丹(Glechoma hederacea L.)营养器官解剖学研究

对欧活血丹营养器官解剖学进行研究,结果表明：欧活血丹营养器官具有双子叶植物的基本特征.根由表皮、皮层、维管柱组成,初生木质部二原型,外始式发育;次生生长时不形成周皮,只形成次生维管组织,次生木质部发达.茎横切面为正方形,由表皮、皮层、维管柱组成;茎的表皮有少量的腺体与表皮毛,4个角隅处有厚角组织分布,厚角组织内侧为4个大维管束,髓发达;上、下叶表皮细胞差异显著,上表皮细胞大,疏被表皮毛,腺点及气孔多分布于下表皮;叶肉栅栏组织发达,海绵组织形态多样、排列疏松;叶为网状脉,主脉维管束发达,侧脉及各个支脉的结构因大小不同而逐渐简化.叶柄横切面为马蹄形,有三个维管束,中央大维管束发达,角隅处小维管束简化且具厚角组织.     

Mechanosensitivity of mammalian auditory hair cells in vitro

Intracellular responses recorded in vitro from the cochleas of anaesthetized mammals have shown that the mechanoreceptive inner and outer hair cells are sharply tuned, accounting for many of the properties of the afferent fibres in the auditory nerve. However, in vivo it has not been possible to measure directly the excitatory mechanical input to these cells (the displacement of their mechanosensitive stereocilia) and thus to determine the relationship between the receptor potentials and displacement of their stereocilia. As a means of circumventing this technical difficulty, we have developed an organ culture of the mouse cochlea and here we describe the receptor potentials generated by the hair cells in response to direct displacement of their stereocilia.     

用无网格法求解不同Re下圆柱绕流问题

采用无网格伽辽金法对不同Re下二维不可压粘性流场圆柱绕流问题进行了数值模拟。该计算不仅预测了不同条件下的流动特性,而且预测了流体作用在圆柱上的曳力和升力。这些力通过积分圆柱表面的压力和粘性力获得。结果显示,当Re大于80时,圆柱所受曳力和升力开始振荡。计算所得的脱落频率(Sr)与前人的结果吻合良好。     

轴向流中两平行弹性薄板大挠度流固耦合系统的数值模拟

研究了轴向流中两平行弹性薄板大挠度流固耦合系统的振动响应和流场特性.板的结构动力学方程采用基于位移的有限元法离散,流场采用完全的二维不可压缩粘性流体N-S方程,用有限体积法离散,结合动网格控制技术,建立了模拟轴向流中两平行悬臂弹性薄板双向流固耦合作用的二维数值模型,实现了弹性薄板—流体—弹性薄板之间的交互作用.利用该数值模型得到了单块板的流致振动特性;发现了随着两板间距大小的不同,两板表现出三种不同的的极限环振动:同相位、异相位以及不确定相位,并得到了同相位与异相位两种振动状态下的板边界层脱落及尾流变化规律;另外,分析还发现在同相位和异相位过渡阶段,两板均表现出拍现象.     

悬浮式磁流体惯性传感黏阻与量程可控机理

实现悬浮式磁流体惯性传感的量程控制关键在于分析基于经典黏性流体力学理论的动态黏度特性和流固系统运动特性.在质量块作简谐运动下,建立惯性传感结构中间隙的流动模型并对理论方程进行求解.在不同动力黏度下,对作简谐运动的传感运动块进行流阻力进行数值计算,结果表明:质量块所受黏阻力主要是两侧压力差,压力差随时间呈现简谐特性,幅值与磁流体动力黏度有较好的线性关系;在选定实验参数后研究结果为压差相位超前22°～40°,剪切应力超前在15°以内,且随着动力黏度增大而减少,当运动黏度达到75 pma·s时,相位超前量变化趋缓.