Regulation of bidirectional transport of the nucleus by adapter proteins

衔接蛋白调节细胞核的双向运输

• 批准号: 10579250
• 负责人: Sozanne R Solmaz
• 金额: $ 35.55万
• 依托单位: STATE UNIVERSITY OF NY,BINGHAMTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10579250
• 关键词: Adaptor Signaling Protein; Affect; Affinity; Amyotrophic Lateral Sclerosis; Binding; Biochemical; Biological Assay; Biological Models; Biophysics; Brain; Calorimetry; Cause of Death; Cell Cycle; Cell Cycle Regulation; Cell Nucleus; Cells; Chromosome Segregation; Collaborations; Complex; Cytoplasm; Data; Defect; Development; Disease; Drosophila genus; Dynein ATPase; Event; G2 Phase; Genetic; Hereditary Spastic Paraplegia; Infant; Kinesin; Length; Link; Methods; Microtubules; Mitotic spindle; Modeling; Molecular; Motor; Muscle; Muscle Development; Mutagenesis; Mutation; NMR Spectroscopy; Neocortex; Neuroglia; Neuromuscular Diseases; Neurons; Neutrons; Nuclear; Nuclear Envelope; Nuclear Pore; Nuclear Pore Complex Proteins; Pathway interactions; Play; Pore Proteins; Positioning Attribute; Process; Proteins; Radial; Registries; Regulation; Resolution; Roentgen Rays; Role; Running; Signal Transduction; Site; Slide; Speed; Spinal Muscular Atrophy; Structure; Synapses; Testing; Titrations; War; X-Ray Crystallography; alpha helix; biophysical techniques; cell motility; cell type; crosslink; dynactin; human disease; improved; insight; interdisciplinary approach; kinetic model; molecular dynamics; mutant; neocortical; novel; reconstitution; recruit; single molecule; spatiotemporal; stem cells; transmission process

PROJECT SUMMARY The cell nucleus is bi-directionally transported and positioned in a cell cycle specific manner, a process that is important for cell cycle control as well as brain and muscle development. The importance of nuclear positioning for brain and muscle development is underscored by the fact that human disease mutations of proteins engaged in the transport of the nucleus cause severe brain and muscle development diseases, including amyotrophic lateral sclerosis (ALS), hereditary spastic paraplegia and spinal muscular atrophy, which is the most common genetic cause of death in infants. Yet, it is unknown how teams of opposing motor complexes collaborate to achieve correct timing, directionality and velocity of transport. Notably, the interactions of opposing motors with cargoes have not been characterized by biochemical or structural methods as proposed here. The nuclear pore complex protein Nup358 provides recruitment sites at the nuclear envelope for the opposing motor complexes dynein and kinesin-1, which can bind simultaneously and facilitate bi-directional positioning of the nucleus along microtubules. This pathway is important for faithful chromosome segregation and essential for a fundamental process in brain development that is required for brain progenitor cells to differentiate to neurons and other cell types. Dynein adapters such as Bicaudal D2 (BicD2) have key roles in transport, as they select cargoes and are required to activate dynein for processive motility; however, the underlying molecular mechanism is unknown. We plan: 1) To establish how dynein motility is modulated by dynein adapter/cargo complexes and by kinesin- 1. 2) to establish a structural basis for recognition of the cell nucleus as cargo by dynein adapters. 3) To establish whether dynein and kinesin-1 are recruited in a cooperative manner to Nup358 at the nucleus, i.e. whether binding of the first motor changes the affinity for the second motor. We plan to establish how BicD2/cargo complexes activate dynein for processive motility. Furthermore we plan to assess how kinesin-1 modulates motility of the dynein/BicD2/Nup358 complex. Our approach combines NMR spectroscopy, X-ray crystallography and biophysical methods, which are integrated with single-molecule processivity assays with intact dynein and kinesin-1 motors. Results will establish a structural basis for cargo selection by BicD2. Our study serves as a model system to understand how cargo adapters regulate the motility and directionality of cargo transported bi-directionally by both dynein and kinesin-1, which is important as these motors facilitate a vast number of cellular transport events that are essential for chromosome segregation, signal transmission at synapses, brain and muscle development. More specifically, results will establish how correctly timed bi-directional transport of the nucleus is regulated, which is crucial for cell cycle control, muscle and brain development. Mutations of proteins of these pathways cause devastating neuromuscular diseases, and results will help devise therapies for these diseases.

项目摘要 细胞核以细胞周期特异性方式双向运输和定位，这是一个 对细胞周期控制以及大脑和肌肉发育都很重要。核定位的重要性 对于大脑和肌肉发育的影响， 从事运输的核造成严重的大脑和肌肉发育疾病，包括 肌萎缩性侧索硬化症（ALS）、遗传性痉挛性截瘫和脊髓性肌萎缩症，这是 最常见的婴儿死亡遗传原因。然而，我们还不知道， 协作以实现正确的时间、方向和运输速度。值得注意的是， 具有货物的对置发动机还没有如所提出的那样通过生物化学或结构方法来表征 这里.核孔复合物蛋白Nup 358在核膜处为细胞核提供募集位点。 相反的运动复合体动力蛋白和驱动蛋白-1，可以同时结合，并促进双向 核沿着微管的定位。这条通路对于染色体的可靠分离是重要的 并且对于大脑发育的基本过程是必不可少的，这是大脑祖细胞所必需的， 分化成神经元和其他细胞类型。动力蛋白衔接子如Bicaudal D2（BicD 2）在免疫调节中具有关键作用。 运输，因为它们选择货物，并需要激活动力蛋白进行运动;然而， 潜在的分子机制是未知的。 我们计划：1）建立动力蛋白运动性是如何通过动力蛋白接头/货物复合物和驱动蛋白调节的， 1. 2)建立一个识别细胞核作为货物的动力蛋白适配器的结构基础。3)到 确定动力蛋白和驱动蛋白-1是否以合作的方式在细胞核处被募集到Nup 358，即 第一马达的结合是否改变对第二马达的亲和性。我们计划确定 BicD 2/货物复合物激活动力蛋白进行性运动。此外，我们计划评估驱动蛋白-1 调节动力蛋白/BicD 2/Nup 358复合物的运动性。 我们的方法结合了NMR光谱学，X射线晶体学和生物物理学方法，这些方法是 与具有完整动力蛋白和驱动蛋白-1马达的单分子持续合成能力测定相结合。结果将 为BicD 2的货物选择建立结构基础。我们的研究作为一个模型系统来理解 货物适配器如何调节由动力蛋白双向运输的货物的运动性和方向性 和驱动蛋白-1，这是重要的，因为这些马达促进了大量的细胞运输事件， 对染色体分离、突触信号传递、大脑和肌肉发育至关重要。更 具体地说，研究结果将确定如何正确地调节细胞核的定时双向运输， 对细胞周期控制、肌肉和大脑发育至关重要。这些途径的蛋白质突变导致 毁灭性的神经肌肉疾病，研究结果将有助于设计这些疾病的治疗方法。