Structural and Molecular Basis of Transduction in Auditory Sensory Organs

听觉感觉器官转导的结构和分子基础

• 批准号: 10916864
• 负责人: BECHARA KACHAR
• 金额: $ 295.76万
• 依托单位: NATIONAL INSTITUTE ON DEAFNESS AND OTHER COMMUNICATION DISORDERS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10916864
• 关键词: Actins; Affect; Aging; Anatomy; Architecture; Auditory; Automobile Driving; Binding; Biological Assay; Cavia; Cell membrane; Cellular Structures; Cilia; Cochlea; Collaborations; Compensation; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Development; Dimensions; Dynein ATPase; Elasticity; Electron Microscopy; Epithelium; Exhibits; Filament; Flagella; Freeze Etching; Freezing; Genes; Goals; Hair; Hair Cells; Image; In Situ; Individual; Inner Hair Cells; Kinocilium; Knock-out; Laboratories; Lateral; Length; Life; Link; Location; Maintenance; Membrane; Messenger RNA; Methodology; Methods; Microscopy; Microtomy; Microtubule Bundle; Molecular; Molecular Conformation; Morphology; Mus; Nature; Organ; Organ of Corti; Outer Hair Cells; Pattern; Phenotype; Physical condensation; Pillar Cell; Positioning Attribute; Process; Prolate; Property; Protein Databases; Protein Family; Proteins; Radial; Rana; Rattus; Regulation; Research; Role; Scanning; Sensory; Sensory Hair; Series; Shapes; Signal Transduction; Source; Stereocilium; Structure; Supporting Cell; System; Testing; Therapeutic Intervention; Tissue Embedding; Transmission Electron Microscopy; Vestibular Hair Cells; absorption; arm; biological systems; cell motility; cell type; cilium motility; cold temperature; crosslink; deafness; design; genetic regulatory protein; hearing impairment; innovation; light microscopy; mRNA Expression; mechanotransduction; nexin; novel; postnatal; preservation; preventive intervention; programs; protein complex; scaffold; self organization; spatiotemporal; tomography

Molecular identity, structure, and elastic properties of the stereocilia tip and lateral links: Elucidating the identity, structure, and elastic properties of the tip and lateral links between stereocilia is essential for understanding their fundamental role in the process of mechanotransduction (MET). Because of their small dimensions, scarcity, and sensitivity to manipulation, the tip and lateral links are notoriously challenging to analyze morphologically. Tip and lateral links were recently examined by cryo-electron tomography (CryoET); however, they had to be splayed out and separated from their natural bundle organization to meet the freezing and imaging requirements for CryoET. We have been able to use freeze-etching combined with transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) tomography to image stereocilia links in situ, preserving both their undisturbed position and intricate conformation. In vestibular hair cells (VHCs) stereocilia bundles, we discovered an elaborate network of radial links crosslinking the hexagonally packed stereocilia within each bundle. Some of these intertwined filaments branched or made lateral contacts with neighboring filaments along the length of the stereocilia, with enrichment in shorter stereocilia. These radial links vary from 120 to 200 nm in length and show extendibility in thin sections of directly frozen and low temperature embedded tissue. Similar links were observed between the kinocilium and the neighboring stereocilia. Notably, these radial links were observed across species, including mouse, rat, and guinea pig, underscoring their significance in the dynamic properties and mechanosensitivity of hair bundles in VHCs. We are currently in the process of reconstructing the tilt series and segmenting all stereocilia links to determine their dimensions, substructure, insertion points, lateral associations, and branching patterns to elucidate their roles in bundle dynamic properties and MET. Molecular identities of components within novel vestigial kinociliary structure in inner and outer hair cells: Cilia and flagella are notable for their motile properties. By contrast, primary cilia, also known as immotile cilia, function mainly as facilitators of signal transduction. Among these various cilia, the kinocilium stands out due to its intriguing functional significance within the organ of Corti and the vestibular system. Positioned adjacent to the tallest stereocilia and connected to the stereocilia bundle, the kinocilium has sparked substantial debate concerning its dynamic properties and role in stereocilia bundle formation and MET. We have previously observed flagellar-like beating in kinocilia of frog semicircular VHCs. This observation prompted us to investigate whether the kinocilium share similarities with motile or non-motile (primary-like) cilia. While VHCs maintain kinocilia throughout life, in cochlear hair cells, kinocilia are present early postnatally but are reabsorbed as the hair cell matures, disappearing around postnatal day 12. Strikingly, using freeze-etching we observed a vestigial kinociliary structure with the ring of proteins that form the ciliary necklace. To identify kinociliary proteins, as well as novel proteins, that make up this vestigial kinociliary structure, we are comparing mRNA expression profiles of cochlear and VHCs in collaboration with David He (Creighton Univ). We have identified expression of 85 proteins associated with cilia, spanning various protein families, including CFAP, Ccdc, LRRC, WDR, DRC, NME, Dnah, Dnal, Dnnaf, IFT, TEKT, and RSPH that make up different ciliary structural features. These proteins exhibit diverse localization within different regions of the cilium, encompassing radial spoke proteins (RSP), outer and inner dynein arms (ODA and IDA), central pair complex (CPC), and Nexin dynein regulatory complex (N-DRC). Several of these proteins are distinct markers of motile cilia across a spectrum of biological systems. A specific protein, known as SPEF1 (or CLAMP), was initially discovered in our laboratory as a component of the microtubule bundle in the pillar cells of the cochlea. Our recent findings indicate a notable enrichment of this protein within VHCs relative to cochlear hair cells. Our investigative strategy revolves around leveraging comprehensive mRNA and protein databases, in combination with an exploration of the freeze-etching and CryoEM architecture of the kinocilium and the vestigial kinociliary structure in mature cochlear hair cells. Molecular mechanisms underlying membrane curvature sensing and remodeling at the stereocilia tips: The tips of stereocilia vary in shape and function depending on their ranking in the hair bundle staircase. The tallest stereocilia have oblate tips, while those in the shorter rows, where the MET channel complex is located, have prolate tips. Stereocilia prolate tips exhibit structural variability, likely reflecting local remodeling of the plasma membrane integrated into the dynamic regulation of the actin core. The molecular mechanisms by which the membrane curvature is sensed and remodeled at the stereocilia tips, however, remain unknown. Recently, the gene encoding the I-BAR protein BAIAP2L2 was associated with stereocilia shape regulation and hearing loss. We have determined that BAIAP2L2 resides in a distinct spatial compartment between the membrane and actin regulatory machinery. Further, using a heterologous co-transfection assay, we find that BAIAP2L2 self-organizes into protein condensates when it binds to components of the stereocilia MET complex and actin regulatory proteins. We propose that BAIAP2L2 forms a novel protein condensate-based scaffold that helps sculpt the membrane at stereocilia prolate tips while integrating MET and actin regulatory protein complexes. TMC4 and TMC5 in the development and function of hair cells and supporting cells: In collaboration with Drs. Jung-Bum Shin and Seham Ebrahim (UVA), we continue to investigate the role of TMC4 and TMC5, which are transiently co-expressed in hair cells and supporting cells of the developing organ of Corti and vestibular sensory epithelia. We find that when knocked out individually, these proteins do not cause any overt phenotype. We are in the process of testing whether these proteins compensate for one another.

直立纤毛尖端和侧链的分子特性、结构和弹性特性：阐明直立纤毛尖端和侧链之间的特性、结构和弹性特性对于理解它们在机械转导（MET）过程中的基本作用至关重要。由于它们的尺寸小，稀缺性和对操作的敏感性，尖端和侧连接在形态学上分析是出了名的具有挑战性。最近通过低温电子断层扫描（CryoET）检查了尖端和侧节；然而，为了满足CryoET的冷冻和成像要求，它们必须被展开并从自然束组织中分离出来。我们已经能够使用冷冻蚀刻结合透射电子显微镜（TEM）和扫描透射电子显微镜（STEM）断层成像原位立体纤毛链接，保留其未受干扰的位置和复杂的构象。在前庭毛细胞（vhc）立体纤毛束中，我们发现了一个精细的径向链接网络，将每个束内六边形排列的立体纤毛交联。这些盘绕在一起的细丝沿着立体纤毛的长度与邻近的细丝发生分支或横向接触，并在较短的立体纤毛中富集。这些径向链接的长度从120纳米到200纳米不等，在直接冷冻和低温包埋组织的薄片中表现出可扩展性。在纤毛和相邻的立体纤毛之间也观察到类似的联系。值得注意的是，包括小鼠、大鼠和豚鼠在内的物种都观察到了这些径向联系，这强调了它们在vhc中毛束的动态特性和机械敏感性中的重要性。我们目前正在重建倾斜序列，并对所有立体纤毛连接进行分割，以确定它们的尺寸、亚结构、插入点、横向关联和分支模式，以阐明它们在束动态特性和MET中的作用。

项目成果

Tip links in hair cells: molecular composition and role in hearing loss.

• DOI: 10.1097/moo.0b013e3283303472
• 发表时间: 2009-10
• 期刊: Current opinion in otolaryngology & head and neck surgery
• 影响因子: 1.6
• 作者: Sakaguchi H;Tokita J;Müller U;Kachar B
• 通讯作者: Kachar B

NMII forms a contractile transcellular sarcomeric network to regulate apical cell junctions and tissue geometry.

• DOI: 10.1016/j.cub.2013.03.039
• 发表时间: 2013-04-22
• 期刊: CURRENT BIOLOGY
• 影响因子: 9.2
• 作者: Ebrahim, Seham;Fujita, Tomoki;Millis, Bryan A.;Kozin, Elliott;Ma, Xuefei;Kawamoto, Sachiyo;Baird, Michelle A.;Davidson, Michael;Yonemura, Shigenobu;Hisa, Yasuo;Conti, Mary Anne;Adelstein, Robert S.;Sakaguchi, Hirofumi;Kachar, Bechara
• 通讯作者: Kachar, Bechara

Evidence for changes in beta- and gamma-actin proportions during inner ear hair cell life.

• DOI: 10.1002/cm.21227
• 发表时间: 2015-06
• 期刊: Cytoskeleton (Hoboken, N.J.)
• 作者: Andrade LR

Regulation of stereocilia length by myosin XVa and whirlin depends on the actin-regulatory protein Eps8.

• DOI: 10.1016/j.cub.2010.12.046
• 发表时间: 2011-01-25
• 期刊: CURRENT BIOLOGY
• 影响因子: 9.2
• 作者: Manor, Uri;Disanza, Andrea;Grati, M'Hamed;Andrade, Leonardo;Lin, Harrison;Di Fiore, Pier Paolo;Scita, Giorgio;Kachar, Bechara
• 通讯作者: Kachar, Bechara

Myosin transcellular networks regulate epithelial apical geometry.

肌球蛋白跨细胞网络调节上皮顶端几何形状。

• DOI: 10.4161/cc.26229
• 发表时间: 2013
• 期刊: Cell cycle (Georgetown, Tex.)
• 作者: Ebrahim,Seham;Kachar,Bechara
• 通讯作者: Kachar,Bechara