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: 10003737
• 负责人: BECHARA KACHAR
• 金额: $ 154.32万
• 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/10003737
• 关键词: Actins; Address; Affect; Appearance; Auditory; Binding; Biological Assay; CD3 Antigens; CRISPR/Cas technology; Cell membrane; Cochlea; Collaborations; Complex; Computer Simulation; Databases; Defect; Development; Distal; Expression Profiling; Filopodia; Financial compensation; Gene Cluster; Gene Targeting; Genes; Genetic Translation; Goals; Growth; Hair; Hair Cells; Health Sciences; Human; Immunofluorescence Immunologic; Inherited; Inner Hair Cells; Integral Membrane Protein; Intercellular Junctions; Knock-in Mouse; Labyrinth; Length; Link; MYO7A gene; Maintenance; Measures; Mediating; Membrane; Messenger RNA; MicroRNAs; Molecular; Molecular Structure; Morphology; Mosaicism; Mus; Mutation; Oregon; Organ; Organ of Corti; PCDH15 gene; Phenotype; Population; Preventive Intervention; Process; Prolate; Protein Isoforms; Proteins; Regulation; Regulatory Pathway; Reporting; Research; Role; Sensory; Site; Structure; System; Testing; Therapeutic Intervention; Transfection; Universities; Vestibular Hair Cells; base; deafness; density; design; early onset; gene product; genetic regulatory protein; hearing impairment; interest; mechanotransduction; organizational structure; polymerization; programs; protein complex; protein expression; repository; scaffold; self-renewal; spatiotemporal; therapeutic miRNA; transcriptome sequencing

The hair cell mechanotransduction (MET) channel complex resides at the tips of the second and shorter rows of stereocilia in the hair bundle, which are both dynamic and prolate. In addition to TMC1 and TMC2, other transmembrane proteins have recently been identified as essential for normal MET, including transmembrane inner ear (TMIE). MET currents were reported to be abolished in the absence of TMIE, and Tmie mutations cause deafness in mice and humans. Yet, the spatiotemporal expression of TMIE in stereocilia during development and its copy number relative to other MET components are not known. To address these questions, we generated CRISPR/Cas9-mediated knockin mice expressing GFP-tagged TMIE. We found that, in organ of Corti hair cells, TMIE localizes along the stereocilia during early bundle development and becomes more restricted to stereocilia tips as the bundle matures. TMIE is highly localized at the MET site in most vestibular hair cells but shows a dispersed localization in a sub-population with shorter hair bundles. Further, we found a large pool of TMIE-GFP in cytoplasmic membrane compartments of hair cells, suggesting continuous traffic and turnover. These findings provide new directions to investigate roles of TMIE in hair cells. Although both TMC1 and TMIE are essential for normal MET function, it is not known if and how they interact prior to targeting the MET site. To determine the extent of any interaction or co-traffic in stereocilia, we crossed mice expressing TMIE-GFP with those expressing TMC1-mCherry and assessed the relative expression profiles of both proteins along stereocilia. We found that TMIE-GFP and TMC1-mCherry co-localize at the MET site at stereocilia-tips of mature hair cells. However, during development TMIE-GFP also showed a broader distribution along the length of stereocilia and the two proteins did not co-localize outside the MET site. To verify their interdependence in mice we crossed Tmie-/- mice with mice expressing TMC1-mCherry and TMC2-GFP and observed that the TMCs failed to localize to stereocilia-tips without TMIE. Conversely, TMIE localization was not affected in stereocilia of Tmc1-/- mice. Thus, although TMIE and TMC1 traffic to the MET site independently of each other, TMIE is essential to stabilize TMC1 and TMC2 locally at the MET site. Analysis of stereocilia bundle morphology in the organ of Corti of mice with mosaic expression of TMC1-mCherry and TMC2-GFP on a Tmc1/:Tmc2/ background revealed that either TMC1 or TMC2 was necessary for normal bundle development, supporting the hypothesis that there is a degree of compensation between the two. Because TMIE is essential for TMC1 and TMC2 localization at the MET site and for normal MET function, we hypothesized that its absence would also affect bundle development. We observed that as late as P7, inner hair cells (IHCs) lacking TMIE had multiple rows of stereocilia in a pyramidal organization, reminiscent of Tmc1/:Tmc2/ bundles. This observation further supports the hypothesis that the processes underlying onset of MET and stereocilia development are interconnected and highlight a link between TMIE and TMC1 and TMC2 expression, MET activity, and stereocilia regulation. Stereocilia-tips where the MET channel complex is located, have prolate tips. However, the molecular basis of membrane curvature sensing and remodeling at stereocilia-tips is not known. Recently the gene encoding the I-BAR protein BAIAP2L2 was reported to be associated with hearing loss. We discovered that BAIAP2L2 localizes to stereocilia prolate tips, and the rise of its expression levels coincides with the onset of MET and the expression of stereocilia actin-regulatory proteins. Strikingly, BAIAP2L2 resides in a distinct spatial compartment, between the membrane and actin-regulatory machinery, providing new exciting evidence for the stratified organization of the protein complex(es) at stereocilia tips. Finally, we found, using a heterologous co-transfection assay, that BAIAP2L2 self-organizes into dense molecular aggregates and binds to multiple components of the stereocilia MET complex, as well as MYO3A/B- and MYO15A-based actin-regulatory proteins. We propose that BAIAP2L2 forms a scaffold that helps sculpt the membrane at stereocilia prolate tips and integrates MET and actin-regulatory protein complexes. We previously showed that MYO3A and MYO3B and their cargo ESPN1 localize to stereocilia distal tips, the sites of actin polymerization. In collaboration with Peter Barr-Gillespie (Oregon Health & Science University), we also identified a second MYO3A/3B cargo protein, ESPNL (espin-like), which appears early in stereocilia development and showed that that ESPN1 and ESPNL interact differently with MYO3A/B to regulate stereocilia staircase step-size. We next generated Espn1-/-:EspnL-/- mice to ascertain the degree of compensation between the two cargo proteins in determining stereocilia staircase morphology. We found that in mice lacking both ESPN1 and ESPNL the growth of the shorter rows of stereocilia begins to slow at P3, and almost all are lost completely by P12. Correspondingly, these mice have severe hearing loss, a complete loss of DPOAEs (as a measure of OHC function), and a 90 % reduction in OHC MET currents. Also, MYO3B, which we previously showed requires ESPN1 or ESPNL to localize to stereocilia-tips, is no longer able to do so. The stereocilia phenotype in mice lacking ESPN1 and/or ESPNL suggests some compensation between them in generating the stereocilia staircase and highlights the complex crosstalk between MYO3A/3B and their cargos in this process. In a previous study we demonstrated that the deletion of the polycistronic miRNA-183 cluster inhibited stereocilia elongation and global bundle maturation. We hypothesize that the miR-183 cluster coordinates the expression of multiple stereocilia actin-regulatory proteins via selective blocking of mRNA translation, and that this regulatory pathway provides an opportunity to design and test miR-based therapeutic approaches to rescue or regrow stereocilia. We used an in-silico approach to predict miR-183 cluster gene targets that overlap with stereocilia-specific mRNAs in the RNA-Seq repository database (gEAR Portal). The gene targets identified include Myo3b, Eps8, Triobp, which are known to be involved in regulation of stereocilia bundle structure and anchoring to the cuticular plate. We are currently using immunofluorescence to examine and quantify the changes in the stereocilia expression of these proteins in absence of the miR183 cluster. We previously showed that the appearance of MYO3A in cochlear stereocilia coincides with the onset of MET, and a recent report suggests that MYO3A is involved in transport of the PCDH15-CD2 isoform. We also observed that in long vestibular stereocilia, MYO7A shows a base-to-tip gradient of distribution, similar to MYO3A, and consistent with tipward translocation and dynamic accumulation. Based on these observations, we hypothesize that MYO3A and MYO7A transport components of the MET complex to stereocilia tips with the potential for complementary function and/or redundancy. Using a heterologous expression system, we observed that while MYO3A transports to filopodia tips only the PCDH15-CD2 isoform, MYO7A can transport all three (CD1, CD2, and CD3) PCDH15 isoforms. In a collaboration with Jung-Bum Shin (UVA), we helped demonstrate that the hair cell protein LIM only protein 7 (LMO7) localizes to the cuticular plate and to hair cell junctions. We showed that LMO7 forms actin networks via two putative actin binding domains and that Lmo7-/- mice suffer multiple cuticular plate deficiencies, including reduced actin density and abnormal stereociliary rootlets. Together, these defects affect cochlear tuning and sensitivity in mice lacking LMO7.

毛细胞机械传导 (MET) 通道复合体位于发束中第二排和较短的静纤毛的尖端，这些静纤毛是动态的且呈长形。除了 TMC1 和 TMC2 之外，其他跨膜蛋白最近被确定为正常 MET 所必需的，包括跨膜内耳 (TMIE)。据报道，如果没有 TMIE，MET 电流就会消失，而 Tmie 突变会导致小鼠和人类耳聋。然而，静纤毛在发育过程中 TMIE 的时空表达及其相对于其他 MET 成分的拷贝数尚不清楚。为了解决这些问题，我们培育了表达 GFP 标记 TMIE 的 CRISPR/Cas9 介导的敲入小鼠。我们发现，在柯蒂毛细胞器官中，TMIE 在早期束发育过程中沿着静纤毛定位，并且随着束成熟而变得更加局限于静纤毛尖端。 TMIE 高度定位于大多数前庭毛细胞的 MET 部位，但在发束较短的亚群中显示出分散定位。此外，我们在毛细胞的细胞质膜区室中发现了大量 TMIE-GFP，表明存在连续的交通和周转。这些发现为研究 TMIE 在毛细胞中的作用提供了新的方向。 尽管 TMC1 和 TMIE 对于正常 MET 功能都是必需的，但尚不清楚它们在靶向 MET 位点之前是否以及如何相互作用。为了确定静纤毛中任何相互作用或共同交通的程度，我们将表达 TMIE-GFP 的小鼠与表达 TMC1-mCherry 的小鼠杂交，并评估两种蛋白沿静纤毛的相对表达谱。我们发现 TMIE-GFP 和 TMC1-mCherry 共定位于成熟毛细胞静纤毛尖端的 MET 位点。然而，在发育过程中，TMIE-GFP 也显示出沿静纤毛长度的更广泛分布，并且两种蛋白质并未共定位于 MET 位点之外。为了验证它们在小鼠中的相互依赖性，我们将 Tmie-/- 小鼠与表达 TMC1-mCherry 和 TMC2-GFP 的小鼠杂交，并观察到在没有 TMIE 的情况下，TMC 无法定位到静纤毛尖端。相反，TMIE 定位在 Tmc1-/- 小鼠的静纤毛中不受影响。因此，尽管 TMIE 和 TMC1 彼此独立地传输到 MET 站点，但 TMIE 对于在 MET 站点本地稳定 TMC1 和 TMC2 至关重要。 对在 Tmc1/:Tmc2/ 背景下表达 TMC1-mCherry 和 TMC2-GFP 的小鼠 Corti 器官中静纤毛束形态的分析表明，TMC1 或 TMC2 对于正常束发育是必需的，支持了两者之间存在一定程度补偿的假设。由于 TMIE 对于 MET 位点的 TMC1 和 TMC2 定位以及正常 MET 功能至关重要，因此我们假设它的缺失也会影响束的发育。我们观察到，晚至 P7，缺乏 TMIE 的内毛细胞 (IHC) 在锥体组织中具有多排静纤毛，让人想起 Tmc1/:Tmc2/ 束。这一观察结果进一步支持了以下假设：MET 发生和静纤毛发育的潜在过程是相互关联的，并强调了 TMIE 与 TMC1 和 TMC2 表达、MET 活性和静纤毛调节之间的联系。 立体纤毛尖端是 MET 通道复合体所在的位置，具有长长的尖端。然而，静纤毛尖端膜曲率传感和重塑的分子基础尚不清楚。最近，据报道编码 I-BAR 蛋白 BAIAP2L2 的基因与听力损失有关。我们发现 BAIAP2L2 定位于静纤毛长形尖端，其表达水平的上升与 MET 的发生和静纤毛肌动蛋白调节蛋白的表达相一致。引人注目的是，BAIAP2L2 位于膜和肌动蛋白调节机制之间的独特空间隔室中，为静纤毛尖端蛋白质复合物的分层组织提供了新的令人兴奋的证据。最后，我们使用异源共转染测定发现，BAIAP2L2 自组织成致密分子聚集体，并与静纤毛 MET 复合物的多个成分以及基于 MYO3A/B 和 MYO15A 的肌动蛋白调节蛋白结合。我们提出 BAIAP2L2 形成一个支架，帮助塑造静纤毛长形尖端的膜，并整合 MET 和肌动蛋白调节蛋白复合物。 我们之前表明，MYO3A 和 MYO3B 及其货物 ESPN1 定位于静纤毛远端，即肌动蛋白聚合的位点。我们与 Peter Barr-Gillespie（俄勒冈健康与科学大学）合作，还鉴定了第二种 MYO3A/3B 货物蛋白 ESPNL（espin 样），它出现在静纤毛发育的早期，并表明 ESPN1 和 ESPNL 与 MYO3A/B 相互作用以不同方式调节静纤毛阶梯步长。接下来，我们生成了 Espn1-/-:EspnL-/- 小鼠，以确定两种货物蛋白在确定静纤毛阶梯形态时的补偿程度。我们发现，在同时缺乏 ESPN1 和 ESPNL 的小鼠中，较短的静纤毛行的生长在 P3 时开始减慢，并且几乎全部在 P12 时完全消失。相应地，这些小鼠有严重的听力损失、DPOAE 完全丧失（作为 OHC 功能的衡量标准），并且 OHC MET 电流减少 90%。此外，我们之前展示的 MYO3B 需要 ESPN1 或 ESPNL 才能定位到静纤毛尖端，但现在不再能够这样做。缺乏 ESPN1 和/或 ESPNL 的小鼠的静纤毛表型表明它们在产生静纤毛阶梯方面存在一定的补偿，并强调了在此过程中 MYO3A/3B 与其货物之间的复杂串扰。 在之前的一项研究中，我们证明多顺反子 miRNA-183 簇的缺失会抑制静纤毛伸长和整体束成熟。我们假设 miR-183 簇通过选择性阻断 mRNA 翻译来协调多种静纤毛肌动蛋白调节蛋白的表达，并且该调节途径为设计和测试基于 miR 的治疗方法来拯救或再生静纤毛提供了机会。我们使用计算机模拟方法来预测与 RNA-Seq 存储数据库 (gEAR Portal) 中静纤毛特异性 mRNA 重叠的 miR-183 簇基因靶标。确定的基因靶标包括 Myo3b、Eps8、Triobp，已知这些基因参与调节静纤毛束结构和锚定至角质板。我们目前正在使用免疫荧光来检查和量化这些蛋白质在没有 miR183 簇的情况下静纤毛表达的变化。 我们之前表明，MYO3A 在耳蜗静纤毛中的出现与 MET 的发生一致，最近的一份报告表明 MYO3A 参与 PCDH15-CD2 亚型的运输。我们还观察到，在长前庭静纤毛中，MYO7A 显示出基部到尖端的分布梯度，与 MYO3A 类似，并且与尖端易位和动态积累一致。基于这些观察，我们假设 MYO3A 和 MYO7A 将 MET 复合物的成分转运到静纤毛尖端，具有互补功能和/或冗余的潜力。使用异源表达系统，我们观察到虽然MYO3A仅将PCDH15-CD2亚型转运至丝状伪足尖端，但MYO7A可以转运所有三种（CD1、CD2和CD3）PCDH15亚型。 在与 Jung-Bum Shin (UVA) 的合作中，我们帮助证明了毛细胞蛋白 LIM only 蛋白 7 (LMO7) 定位于角质板和毛细胞连接处。我们发现 LMO7 通过两个假定的肌动蛋白结合域形成肌动蛋白网络，并且 Lmo7-/- 小鼠患有多种角质板缺陷，包括肌动蛋白密度降低和异常的静纤毛细根。这些缺陷共同影响缺乏 LMO7 的小鼠的耳蜗调谐和敏感性。