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。核孔复合体(NPC)作为 NE上分子交换的主要通道，而核-和 细胞骨架(LINC)复合体是跨越NE的物理纽带。Lincs是定位所必需的 在一系列不同的情况下，用于机械传感。这台机器出了故障 人类重要疾病的核心，包括骨骼和心脏肌病、过早衰老和癌症。我们的 目的是了解参与核质通讯的蛋白质复合体的结构。 高(原子)分辨率。这样的信息有助于识别和分离这台机器的无数功能 这一点我们才刚刚开始完全掌握。高分辨率信息进一步提供了 结构导向药物设计的基础，以干扰诸如Emery-Dreifuss等突出的人类疾病 肌营养不良症(EDMD)和原发性肌营养不良症，至今仍未治愈。化合物的结构特征 NPC和LINC综合体具有挑战性，因为这些多MDA的规模和复杂性 装配。在过去的15年里，我们在这两个问题上都取得了重大进展。对于全国人大来说，我们 选择了一种高效的自下而上的方法，在这种方法中，我们表征了多亚单位复合体 主要是通过X射线结晶学，构建巨大的，40-100丙二醛鼻咽癌。那些建筑 现已与装配的NPC的冷冻电子断层扫描(Cryo-ET)图结合使用，以 生成的复合结构可以暂时定位一个NPC中的大约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分量，并开始解开 其组件的不同网络，SAD1/UNC-84(SUN)和Klarsicht/ANC1/Syne-Homology(KASH) 蛋白质。展望未来，挑战是大型和动态装配的结构特征，这是 对于NPC和LINC建筑群都是如此，对于后者来说，特别是当包括与 核素和细胞骨架成分。近年来，低温电子显微镜(Cryo-EM)取得了显著进展 最近的过去使这项技术对我们的研究尤为重要。我们预计会结合X射线 结晶学和低温电子显微镜，用于研究未来最相关的结构。这一意愿的成功 依靠创新的方法来应对每个项目带来的特殊挑战。我们有 反复展示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. Nuclear pore complexes (NPCs) serve as the main conduit for molecular exchange across the NE, while universally conserved linker of nucleo- and cytoskeleton (LINC) complexes serve as physical tethers across the NE. LINCs 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 can tentatively 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 methods to address the particular challenges that come with each project. We have repeatedly shown how to successfully approach such challenges and have devised methods to meet them.