Motor properties and regulation of human myosin 3B

人肌球蛋白 3B 的运动特性和调节

• 批准号: 10371409
• 负责人: Laura Kay Gunther
• 金额: $ 1.47万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-06-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10371409
• 关键词: ATP phosphohydrolase; Actin-Binding Protein; Actins; Affinity; Binding; Biochemical; Biological; Biological Assay; Biophysics; Brain; Bundling; COS-7 Cell; Calcium; Cell physiology; Cells; Collaborations; Complement; Cultured Cells; Dependence; Disease; Drosophila genus; Foundations; Future; Genes; Goals; Hair Cells; Hearing; Hela Cells; Human; Impairment; In Vitro; Intestines; Kidney; Kinetics; Labyrinth; Lead; Length; Molecular; Molecular Motors; Motor; Motor Activity; Mutation; Myosin ATPase; Myosin III; N-terminal; Phosphotransferases; Photoreceptors; Physiological; Property; Protein Isoforms; Proteins; Regulation; Research Project Grants; Role; Sensory; Structure; Tail; Testis; United States National Institutes of Health; Vertebrate Photoreceptors; Vertebrates; base; biophysical analysis; cell motility; deafness; early onset; in vitro Assay; in vivo; insight; novel; single molecule

Abstract: The objective of this proposal is to better understand the properties of Myo3B and its role as a transporter in actin bundle based structures such as the stereocilia of inner ear hair cells. Class III myosins are known actin based motors and are unique because they contain an N-terminal kinase domain that serves to autoregulate motor activity. In vertebrates there exists two class III myosin isoforms; Myo3A and Myo3B. Class III myosins are expressed in inner ear hair cells, photoreceptors, brain, testis, and intestines. Both Myo3A and Myo3B have been shown to bind the actin bundling protein Espin and transport it to the tips of actin protrusions to promote elongation. Myo3A harbors deafness associated mutations while Myo3B is proposed to partially compensate for lack of functional Myo3A, which results in delayed onset deafness. In addition, we recently discovered that Myo3B is strongly regulated by physiological concentrations of calcium, while Myo3A is not. Therefore, it is crucial to characterize the intrinsic motor properties of the Myo3B motor and understand how it is calcium regulated to determine its function in parallel actin based structures. We will investigate the calcium regulation of Myo3B with in vitro assays such as steady-state ATPase, in vitro motility, and transient kinetic analysis with purified proteins which will allow us to determine the intrinsic motor properties of MYO3B. In addition, cell biological studies using cultured cells will enable us to examine the calcium regulation of Myo3B localization and impact on actin protrusion dynamics. Overall, this study will provide fundamental information about the motor mechanism of Myo3B, which will reveal crucial information about its function in the stereocilia of inner ear hair cells.

摘要： 这项建议的目的是更好地了解Myo3B的属性及其作为一种 肌动蛋白束结构中的转运蛋白，如内耳毛细胞的纤毛。第III类 肌球蛋白是已知的以肌动蛋白为基础的马达，它是独特的，因为它们含有一个N-末端的激酶 用于自动调节运动活动的域。脊椎动物中存在两个III类肌球蛋白 亚型：Myo3A和Myo3B。III类肌球蛋白在内耳毛细胞、光感受器、 大脑、睾丸和肠道。Myo3A和Myo3B都被证明与肌动蛋白束结合 蛋白质旋转，并将其运输到肌动蛋白突起的尖端，以促进伸长。Myo3A港湾 耳聋相关突变而Myo3B被认为是部分弥补功能缺失的 Myo3A，这会导致迟发性耳聋。此外，我们最近发现Myo3B是 受生理浓度的钙的强烈调节，而Myo3A则不是。因此，它是 对表征Myo3B马达的固有马达特性并了解其如何 钙调节以决定其在平行肌动蛋白基础结构中的功能。我们将调查 用稳态ATPase、体外运动和钙调节等方法研究Myo3B对钙离子的调节 用纯化的蛋白质进行瞬时动力学分析，这将使我们能够确定内在马达 MYO3B的性质。此外，使用培养细胞进行的细胞生物学研究将使我们能够检查 Myo3B定位的钙调节及其对肌动蛋白突起动力学的影响。总体而言，这 研究将提供有关Myo3B运动机制的基本信息，这将揭示 关于它在内耳毛细胞的立体纤毛中的作用的关键信息。