Mechanics of Inner Ear Hair Bundles

内耳毛束的力学

• 批准号: 8011050
• 负责人: Kiriaki Domenica Karavitaki
• 金额: $ 16.41万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8011050
• 关键词: Adhesions; Antibodies; Area; Binding; Calcium; Carbon Nanotubes; Cellular biology; Chelating Agents; Chemicals; Chemistry; Cochlea; Communities; Confocal Microscopy; Coupled; Coupling; Custom; Dimensions; Discontinuous Capillary; Enzymes; Fourier Analysis; Frequencies; Glycocalyx; Glycoproteins; Goals; Hair; Hair Cells; Hearing; Human; Image; Individual; Inherited; Kinocilium; Label; Labyrinth; Lectin; Link; Measurement; Mechanics; Mediating; Membrane; Microfabrication; Microscope; Modeling; Motion; Movement; Mus; Mutation; Nanotubes; Optics; Outer Hair Cells; Physiology; Proteins; Rana catesbeiana; Relative (related person); Resolution; Resources; Saccule structure; Shapes; Silicon; Slide; Sorting - Cell Movement; Stereocilium; Stimulus; Surface; Technology; Testing; Time; Work; base; cohesion; deafness; imaging probe; public health relevance; research study; sound; two-photon

DESCRIPTION (provided by applicant): The overall goal is to understand how sound is coupled to individual stereocilia within hair bundles of mammalian cochlea. Previous experiments have been limited by poor stimulus-probe coupling to bundles, which produces stimuli that are inhomogeneous in time and amplitude across stereocilia. Here, I propose to use silicon microfabrication and carbon nanotube technologies, together with new nanotube chemistries, to develop custom-fitted cochlear stimulus probes. These probes will be used to investigate stereocilia bundle mechanics, and (in subsequent projects) the micromechanics of transduction in cochlear hair cells. The project will also create a resource for the cochlear physiology community. In one aim, the anatomical dimensions of the V-shaped inner and outer hair cell hair bundles in the mouse cochlea will be quantified by labeling their membranes with FM1- 43 and visualizing them using confocal microscopy. Using these measurements a silicon based V-shaped stimulation probe will be microfabricated and its tip will be coated with carbon nanotubes (CNTs) to promote adhesion of the probe to the cochlear bundles. Adhesion will be tested in FM-143 labeled mouse cochlear explants by attaching the probe to the bundle, displacing the probe and imaging the motion of the bundle under a confocal microscope. If necessary, CNTs will be coated with glycoproteins or stereociliaspecific antibodies to promote adhesion. In the second aim, the V-shaped stimulus probe will be used to quantify the displacement of individual stereocilia within the inner and outer cochlear hair bundles in the mouse cochlea. The probe will be driven by a piezoelectric actuator using varying frequency sinusoids. The motion will be observed using a two photon microscope and DIC optics and quantified by applying Fourier analysis on the captured images. The same experiments will be repeated after applying the calcium chelator BAPTA to cut the tip links. The resulting measurements will help us understand the contribution of the tip links to hair bundle mechanics. PUBLIC HEALTH RELEVANCE: These experiments have two goals: First, by understanding how the stereocilia move together in a mammalian hair cell, we can generate quantitative biophysical models for how hearing works in the kilohertz range. Second, this will help identify the links that permit sliding adhesion of stereocilia. Mutation of similar links is known to cause inherited deafness in mice and humans, and this sort of sliding adhesion represents a new type of mechanism in cell biology.

描述（由申请人提供）：总体目标是了解哺乳动物耳蜗头发束中的声音与单个立体胶质的耦合。先前的实验受到刺激探针耦合到束的不良限制，束会产生刺激，这些刺激在整个立体尾层中的时间和振幅不均匀。在这里，我建议使用硅微结构和碳纳米管技术，以及新的纳米管化学物质，以开发定制的人工耳蜗刺激探针。这些探针将用于研究立体束机械师，（在随后的项目中）耳蜗毛细胞的转导的微观力学。该项目还将为人工耳蜗生理社区创建资源。在一个目的中，通过用FM1-43标记其膜，并使用共聚焦显微镜将其标记为膜，从而量化了小鼠耳蜗中V形内部和外毛的毛发束的解剖尺寸。使用这些测量值，将对基于硅的V形刺激探针进行微分化，并将其尖端与碳纳米管（CNT）涂覆，以促进探针粘附到耳蜗束上。通过将探针连接到束上，将探针连接到探针并在共聚焦显微镜下置换并成像束运动，将在FM-143标记的小鼠人工耳蜗上测试粘附。如有必要，CNT将涂有糖蛋白或立体特异性抗体，以促进粘附。在第二个目标中，V形刺激探针将用于量化小鼠耳蜗内部和外耳发束内单个立体胶质的位移。该探针将由使用不同频率正弦的压电执行器驱动。将使用两个光子显微镜和DIC光学器件观察该运动，并通过对捕获的图像应用傅立叶分析来量化。应用钙螯合剂BAPTA切割尖端链接后，将重复相同的实验。由此产生的测量将有助于我们了解尖端链接到发束力学的贡献。 公共卫生相关性：这些实验有两个目标：首先，通过了解立体胶体如何在哺乳动物毛细胞中一起移动，我们可以生成定量的生物物理模型，以实现kilohertz范围内听力的工作方式。其次，这将有助于确定允许立体胶体粘附的链接。已知相似联系的突变引起小鼠和人类的遗传性耳聋，这种滑动粘附代表了细胞生物学中的一种新型机制。