Administrative Supplement for Award 93818 (R01GM144578)

项目总结 本补充申请了家长拨款(GM144578)的Vitbot 5直插式冷冻机，这将允许申请 一种新的方法，冷冻电子显微镜(Cryo-EM)，它研究了细胞核是如何 以细胞周期特定的方式双向运输和定位，这一过程对细胞很重要 周期控制以及大脑和肌肉的发育。核定位对大脑和 肌肉发育得到强调的事实是，人类疾病参与蛋白质突变的 核的运输会导致严重的大脑和肌肉发育疾病，包括脊椎肌肉 萎缩，这是导致婴儿死亡的最常见的遗传原因。然而，目前还不清楚对立的球队如何 运动复合体相互协作，以实现正确的时间、方向性和运输速度。 我们计划：1)建立动力蛋白复合体动力蛋白的运动性是如何被动力蛋白适配器/货物调节的 复合体和由相反极性的马达复合体Kinesin-1。2)建立承认的结构基础 通过动力蛋白接头将细胞核作为货物运输出去。3)确定相反的极性马达是否动态和 Kinesin-1以合作的方式被招募到细胞核。 冷冻-EM已在母公司赠款中作为结构确定的替代方法提出，并 将使PI能够将她的结构研究扩展到更大的复合体，包括全长动力蛋白适配器 蛋白质和完整的运动复合体。在目前的方法中，结合了X射线结晶学和核磁共振 对于结构确定，等电点仅限于截断的最小络合物的结构研究 碎片。冷冻-EM将增加父赠款的所有三个目标的影响，因为结构完整 与截断的复合体相比，复合体将揭示更高水平的机械洞察力。这是 对于计划建立激活和货物识别的拟议目标尤其重要 BicD2的机制，以及相反极性的马达如何双向运输kinesin-1和dynein 是由接头蛋白调节的。 我们的研究作为一个模型系统来理解货物转接器是如何调节 由动力蛋白和动蛋白-1双向运输的货物，这一点很重要，因为这些马达有助于 大量对染色体分离至关重要的细胞运输事件，信号在 突触、大脑和肌肉发育。更具体地说，结果将确定如何正确地选择时间间隔。 细胞核的定向运输受到调控，这对细胞周期控制、肌肉和大脑至关重要 发展。这些途径的蛋白质突变会导致毁灭性的神经肌肉疾病，结果是 将有助于设计这些疾病的治疗方法。

PROJECT SUMMARY This supplement requests a Vitrobot 5 plunge freezer for the parent grant (GM144578), which will allow to apply a new approach, cryo-electron microscopy (cryo-EM) to the parent grant, which investigates how 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 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. We plan: 1) To establish how motility of the motor complex dynein is modulated by dynein adapter/cargo complexes and by the opposite polarity motor complex kinesin-1. 2) to establish a structural basis for recognition of the cell nucleus as cargo by dynein adapters. 3) To establish whether the opposite polarity motors dynein and kinesin-1 are recruited in a cooperative manner to the nucleus. Cryo-EM was already proposed in the parent grant as an alternative approach for structure determination and will enable the PI to expand her structural studies to larger complexes including full-length dynein adapter proteins and intact motor complexes. With the current approach, that combines X-ray crystallography and NMR for structure determination, the PI has been limited to structural studies of minimal complexes with truncated fragments. Cryo-EM will increase the impact of all three aims of the parent grant since structures of intact complexes will reveal a greater level of mechanistic insights compared to truncated complexes. This is particularly important for the proposed aims which plan to establish the activation and cargo-recognition mechanism of BicD2, as well as how bi-directional transport of the opposite polarity motors kinesin-1 and dynein is regulated by the adapter proteins. 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.