Structure-Function of Nucleo-Cytoplasmic Communication

项目概要/摘要 真核细胞由它们的细胞器定义，细胞器是膜封闭的隔室，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， 基因转录与蛋白质翻译的分离。由于核被膜（NE）是一个紧密的屏障， 包围细胞核的细胞需要机制来建立和控制核质通讯。 这一机制有两个主要不同的组成部分。核孔复合物（NPC）作为 跨NE分子交换的主要管道，而普遍保守的核和核连接子 细胞骨架（LINC）复合物充当穿过NE的物理系链。LINC是定位所必需的 在不同的环境下，细胞核和机械感应。机器的故障是在 核心的重要人类疾病，包括骨骼和心脏肌病，过早衰老和癌症。我们 目的是了解参与核质通讯的蛋白质复合物的结构， 高（原子）分辨率。这些信息有助于识别和分离这一机制的无数功能 这一点，我们才刚刚开始全面了解。高分辨率信息进一步提供了 结构导向药物设计的基础，以干扰突出的人类疾病，如Emery-Dreifuss 肌营养不良症（EDMD）和原发性肌张力障碍，这仍然没有治愈。的结构表征 由于这些多个多学科设计领域（multi-MDa）的规模和复杂性，NPC和LINC复合体是具有挑战性的 组件.过去15年来，我们在这两个问题上都取得了重大进展。对于NPC，我们 我选择了一种高效的自下而上的方法，在这种方法中，我们描述了多亚基复合物的特征， 主要是通过X射线晶体学，巨大的，40-100 MDa NPC的构建块。这些结构 现在已经与组装NPC的低温电子断层扫描（cryo-ET）图结合使用， 生成复合结构，可以在一个NPC中暂时定位大约500个蛋白质。 对于LINC复合体，我们解决了普遍保守的核心组件，并开始解开 其组成部分的多样化网络，Sad 1/ANC-84（SUN）和Klarsicht/ANC 1/Syne-Homology（KASH） proteins.展望未来，挑战是大型动态组件的结构表征， 对于NPC和LINC复合体两者都是如此，对于后者，特别是当包括到 核和细胞骨架成分。冷冻电子显微镜（cryo-EM）在过去的几年中取得了巨大的进展， 最近的过去使这项技术对我们的研究特别重要。我们预计结合X光 晶体学和冷冻电镜来研究未来最相关的结构。这一举措的成功将 依靠创新的方法来解决每个项目带来的特殊挑战。我们有 我们反复展示了如何成功地应对这些挑战，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. 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.