Structure-Function of Nucleo-Cytoplasmic Communication

项目概要/摘要 真核细胞由其细胞器、膜封闭的隔室来定义，其中特定的细胞 过程进行。细胞核是最大的细胞器，包含所有遗传物质，使 将基因转录与蛋白质翻译分离。由于核膜 (NE) 充当紧密屏障 细胞包围着细胞核，需要机制来建立和控制核-细胞质通讯。 该机器有两个主要不同的组件。一方面，核孔复合物 （NPC）是跨 NE 分子交换的主要渠道。另一方面，普遍 核糖体和细胞骨架（LINC）复合物的保守连接体充当横跨 NE 的物理系链， 对于在不同的情况下定位细胞核和进行机械传感是必要的。 机械功能障碍是重要人类疾病的核心，包括骨骼和心脏疾病 肌病、过早衰老和癌症。我们的目标是了解蛋白质复合物的结构 参与高（原子）分辨率的核-细胞质通讯。此类信息有助于识别 并将这个机器所执行的无数功能分开，而我们才刚刚开始完全掌握这些功能。 高分辨率信息进一步为结构引导药物设计提供基础，以干扰 显着的人类疾病，例如埃默里-德莱福斯肌营养不良症 (EDMD) 和原发性肌张力障碍， 仍未治愈。 NPC 和 LINC 复合体的结构表征具有挑战性，因为 这些多 MDa 程序集的大小和复杂性。过去15年，我们取得了重大成就 在这两个问题上都取得了进展。对于 NPC，我们选择了一种高效的自下而上的方法，其中 我们主要通过 X 射线晶体学来表征多亚基复合物，这是 大型 40-100 MDa NPC。这些结构现已与低温电子结合使用 组装 NPC 的断层扫描（冷冻 ET）图，以生成复合结构，试图定位 一个 NPC 内大约有 500 个单独的蛋白质。对于LINC复合体，我们解决了普遍保守的问题 核心组件，并已开始理清其组件的多样化网络，即 Sad1/UNC-84 (SUN) 和 Klarsicht/ANC1/Syne 同源 (KASH) 蛋白。展望未来，挑战在于结构性 大型动态组件的表征，对于 NPC 和 LINC 复合体都是如此，对于 后者特别是当包括与核和细胞骨架成分的连接时。戏剧性的 最近冷冻电子显微镜 (cryo-EM) 的进步使这项技术特别重要 对我们的学习很重要。我们预计将 X 射线晶体学和冷冻电镜相结合来研究最 相关结构正在推进。这项工作的成功将取决于创新的、量身定制的方法 解决每个项目带来的特殊挑战。过去我们已经多次展示 十年来如何成功应对这些挑战并设计了应对这些挑战的方法。

PROJECT SUMMARY / ABSTRACT Eukaryotic cells are defined by their organelles, membrane-enclosed compartments in which specific cellular processes are carried out. The nucleus is the largest organelle, contains all genetic material, and enables separation of gene transcription from protein translation. As the nuclear envelope (NE) serves as a tight barrier enclosing the nucleus, the cell requires machinery to establish and control nucleo-cytoplasmic communication. There are two principally different components to this machinery. On one hand, nuclear pore complexes (NPCs) serve as the main conduit for molecular exchange across the NE. On the other hand, universally conserved linker of nucleo- and cytoskeleton (LINC) complexes serve as physical tethers across the NE, which are necessary for positioning the nucleus and for mechano-sensing in a diverse set of circumstances. Dysfunction of the machinery is at the core of important human diseases, including skeletal and cardiac myopathies, premature aging, and cancer. Our goal is to understand the structure of the protein complexes involved in nucleo-cytoplasmic communication at high (atomic) resolution. Such information helps to identify and separate the myriad functions this machinery carries out and that we are still only beginning to fully grasp. High resolution information further provides the basis for structure-guided drug design to interfere with the salient human diseases, such as Emery-Dreifuss Muscular Dystrophy (EDMD) and Primary Dystonia, which are still not cured. The structural characterization of the NPC and the LINC complex are challenging, because of the size and complexity of these multi-MDa assemblies. Over the past 15 years, we have made significant advances on both problems. For the NPC, we have chosen a highly productive bottom-up approach, in which we characterized multi-subunit complexes predominantly by X-ray crystallography, the building blocks of the massive, 40-100 MDa NPC. Those structures have now been used in combination with cryo-electron tomographic (cryo-ET) maps of assembled NPCs to generate composite structures that attempt to position the roughly 500 individual proteins within one NPC. For the LINC complex, we solved the universally conserved core component and have started to untangle the diverse network of its components, the Sad1/UNC-84 (SUN) and Klarsicht/ANC1/Syne-Homology (KASH) proteins. Going forward, the challenge is the structural characterization of large and dynamic assemblies, which is true for both, the NPC and the LINC complex, for the latter particularly when including the connection to the nucleo- and cytoskeletal components. The dramatic advances in cryo-electron microscopy (cryo-EM) over the recent past make this technology particularly important for our studies. We anticipate combining X-ray crystallography and cryo-EM for studying the most relevant structures going forward. The success of this will depend upon innovative, tailored methods to address the particular challenges that come with each project. We have repeatedly shown over the past decade how to successfully approach such challenges and have devised methods to meet them.