Structure-Function of Nucleo-Cytoplasmic Communication

项目摘要/摘要 真核细胞是由细胞器定义的，细胞器是由膜包裹的隔间，在其中特定的细胞 过程被执行。细胞核是最大的细胞器，包含所有遗传物质，并使 分离基因转录和蛋白质翻译。因为核包膜(NE)是一道严密的屏障 包围细胞核的细胞需要机械来建立和控制核质通讯。 这台机器有两个主要不同的部件。一方面，核孔复合体 (Npc)是跨越NE的分子交换的主要通道。另一方面，普遍的 保守的核和细胞骨架(LINC)复合体连接物是跨越NE的物理纽带，它 是定位原子核和在不同环境下进行机械传感所必需的。 机械功能障碍是人类重要疾病的核心，包括骨骼和心脏 肌病、早衰和癌症。我们的目标是了解蛋白质复合体的结构 参与高(原子)分辨率的核质通讯。这样的信息有助于识别 并分离这个机器执行的无数功能，而我们仍然只是刚刚开始完全掌握这些功能。 高分辨率信息进一步为结构导向药物设计提供了干扰 人类显著的疾病，如埃默里-德雷弗斯肌营养不良症(EDMD)和原发性肌营养不良症，这些疾病 仍然没有治愈。NPC和LINC复合体的结构特征具有挑战性，因为 这些多MDA程序集的大小和复杂性。在过去的15年里，我们取得了重大成就 这两个问题都取得了进展。对于全国人大，我们选择了一种富有成效的自下而上的方法，在这种方法中 我们主要通过X射线结晶学来表征多亚单位络合物，这是 体型庞大，40-100丙二醛鼻咽癌。这些结构现在已经与冷冻电子结合使用。 装配的NPC的层析(冷冻-ET)图，以生成试图定位 一个鼻咽癌中大约有500个单独的蛋白质。对于LINC复合体，我们求解了普遍守恒的 核心组件，并已开始理清其组件SAD1/UNC-84(SUN)的多样化网络 和Klarsicht/ANC1/Syne-Homology(Kash)蛋白。展望未来，挑战是结构性的 描述大型和动态装配的特征，这对NPC和LINC综合体都适用， 后者尤其是当包括与核和细胞骨架组件的连接时。戏剧性的 近年来低温电子显微镜(Cryo-EM)的进步使这项技术变得尤为重要 对我们的学习很重要。我们期待着将X射线结晶学和低温电子显微镜相结合，以研究最 未来的相关结构。这一成功将取决于创新的、量身定做的方法来 应对每个项目带来的特殊挑战。在过去的一段时间里，我们反复表明 十年来，我们了解了如何成功应对这些挑战，并制定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.