Molecular Mechanisms of Hair Bundle Development and Maintenance

发束发育和维护的分子机制

• 批准号: 10205022
• 负责人: Jonathan Edward Bird
• 金额: $ 39.12万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10205022
• 关键词: ATP phosphohydrolase; ATPase Domain; Actins; Affect; Animals; Architecture; Auditory; Binding; Biochemical; Biological Assay; C-terminal; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Cochlea; Complex; Critical Pathways; Cryoelectron Microscopy; Cytoskeleton; Data; Defect; Detection; Development; EPS8 gene; Ear; Electron Microscopy; Filament; Fluorescence Microscopy; Foundations; GNAI3 gene; GPSM2 gene; Genes; Genetic; Genetic Engineering; Goals; Growth; Hair; Hair Cells; Hearing; Heart; Human; In Vitro; Individual; Inherited; Laboratories; Labyrinth; Life; Longevity; Maintenance; Measures; Mechanics; Microfilaments; Microscopy; Molecular; Molecular Motors; Motor; Mus; Mutant Strains Mice; Mutation; Myosin ATPase; N-terminal; Organelles; Outcome; Pathologic; Pathology; Pathway interactions; Patients; Phase; Process; Property; Protein Isoforms; Proteins; Pyrenes; Resolution; Sensory; Spectrum Analysis; Stereocilium; Structure; Tail; Testing; WHRN gene; Work; deafness; disability; experimental study; hearing impairment; hereditary hearing loss; in vitro activity; in vivo; light microscopy; mechanotransduction; monomer; mouse model; mutant; novel; particle; polymerization; postnatal; preservation; protein complex; single molecule; sound; synergism; trafficking

Project Summary The detection of sound in the cochlea requires hair cells and their mechano-sensitive organelles, called stereocilia. The long-term goal of this laboratory is to study how stereocilia grow and how their integrity is maintained over a lifetime. These are critical processes and are commonly disrupted in hereditary forms of human hearing loss. In this proposal, we investigate a molecular motor called myosin 15 (MYO15A) that sets the size of the actin filament core that is the structural foundation within each stereocilium. Mutations in the MYO15A gene cause human hereditary hearing loss, DFNB3. Our initial experiments have revealed a novel mechanism that allows MYO15A to control the actin core, and we hypothesize that the hair cell regulates stereocilia architecture using different MYO15A isoforms. To test this, we will investigate the molecular properties of MYO15A to understand how it influences growth of the actin core, reveal how these activities are regulated within the hair cell, and examine how mutations cause hearing loss in a mouse model. In Aim 1, we use purified proteins and spectroscopy / single-molecule assays to extensively characterize how MYO15A accelerates actin polymerization. As part of this, we will introduce mutations to explore candidate regions within MYO15A that underlie this activity. In Aim 2, we expand our study to different isoforms of MYO15A and use biochemical assays and cryo-electron microscopy to investigate key differences in their enzymatic activity and how these are regulated. In Aim 3, we characterize a mutant mouse where a novel MYO15A isoform has been removed using CRISPR genetic engineering, and study how these animals lose their hearing using a combination of high- resolution electron and light microscopy. Overall, our proposal will provide critical new information into basic mechanisms of stereocilia plasticity, in addition to revealing the distinct pathologies that cause deafness in patients suffering with DFNB3.

项目总结