Regulation of bidirectional transport of the nucleus by adapter proteins

项目总结 细胞核以细胞周期特定的方式双向运输和定位，这一过程是 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 对于大脑和肌肉的发育，强调了这样一个事实，即人类疾病蛋白质的突变 参与核的运输会导致严重的大脑和肌肉发育疾病，包括 肌萎缩侧索硬化症(ALS)，遗传性痉挛性截瘫和脊肌萎缩，这是 婴儿死亡最常见的遗传原因。然而，目前还不清楚相互对立的运动型复合体是如何 合作以实现正确的时间、方向和速度的运输。值得注意的是， 与货物相反的马达尚未按照建议的生物化学或结构方法进行表征 这里。核孔复合体蛋白Nup358为核膜提供募集部位 相互对立的运动复合体dynein和kinesin-1，可同时结合并促进双向 细胞核沿微管的定位。这条途径对于忠实的染色体分离很重要。 对于大脑发育中的一个基本过程是必不可少的，这是脑祖细胞 分化为神经元和其他类型的细胞。动力蛋白适配器如Bicaudal D2(BicD2)在 运输，因为他们选择货物，并需要激活动力蛋白进行前进运动；然而， 潜在的分子机制尚不清楚。 我们计划：1)建立动力蛋白运动是如何被动力蛋白适配器/货物复合体和动力蛋白- 1.2)建立动力蛋白接头识别细胞核为货物的结构基础。3)至 确定dynein和kinesin-1是否以合作的方式在细胞核内被招募到Nup358，即 第一马达的结合是否改变了第二马达的亲和力。我们计划确定如何 BicD2/Cargo复合体激活Dynein的前进运动。此外，我们计划评估Kinesin-1是如何 调节dynein/BicD2/Nup358复合体的运动。 我们的方法结合了核磁共振光谱、X射线结晶学和生物物理方法，这些方法是 与完整的动力蛋白和动力蛋白-1马达的单分子加工性分析相结合。结果将 为BicD2选择货物奠定结构基础。我们的研究作为一个模型系统来理解 货物转接器如何调节双向运输货物的运动性和方向性 和Kinesin-1，这一点很重要，因为这些马达促进了大量的细胞运输事件，这些事件是 对染色体分离、突触信号传递、大脑和肌肉发育至关重要。更多 具体地说，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会导致 毁灭性的神经肌肉疾病，结果将有助于设计这些疾病的治疗方法。

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.