Structure-Function of Nucleo-Cytoplasmic Communication

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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.