NPCcryoET · A combined approach of cryo-electron tomography and protein tagging for elucidating the structural organization of the human Nuclear Pore Complex
FP7 — People (Marie Curie Actions)
- Duration
- 2012-12-01 → 2014-11-30
- EU contribution
- €174,475
- Participants
- 1
- Scheme
- MC-IEF
Lines connect the coordinator with its partners.
Results in brief
A combined approach of cryo-electron tomography and protein tagging for elucidating the structural organization of the human Nuclear Pore Complex
The project goal is to reveal the structure and organization of the nuclear pore complexes (NPCs), the exclusive gateway between the nucleus and cytoplasm. My proposed research plan is to use a combination of state-of-the-art cryo-electron tomography and subtomogram averaging, and localization of nucleoporins or NPC subcomplexes to reveal the molecular architecture of the NPC. During the project period, I worked to optimize subcellular fractionation protocols for intact nuclear envelopes isolated from Hela cells and cryo-preparation protocol of the isolated nuclear envelopes for cryo-electron tomography. This resulted in an improved and consistent protocol for nuclear envelope isolation and thin vitrified electron microscopy grids for cryo-tilt series acquisition. Furthermore, I have developed a high throughput acquisition workflow for tilt series, improved and developed a subtomogram averaging workflow to obtain the high-resolution structure of the NPC. Together with my colleagues in the host laboratory, I have designed oligos for gene-silencing of several nucleoporins, cloned and created inducible stable-cell lines for the study of the NPC organization in conjunction with several colleagues in the host laboratory. As a result, I was able to obtain the structure of the human NPC at ~32 Angstrom resolution. In parallel, the structure of the NPC from a stable cell line inducibly expressing miRNA targeting the Nup214, a transport nucleoporin was obtained at 40 Å resolution. The comparison between wild type and this structure indicates the localization the Nup214 subcomplex in the NPC. Together with Alexander von Appen, a PhD student in the lab, I reconstructed the negative stained structure of the scaffold human Nup107 subcomplex purified by affinity-pulldown. Fitting this isolated structure of Nup107 subcomplex into the tomographic map of the NPC revealed that 32 copies of the Nup107 subcomplex assemble into two reticulated rings, one each at the cytoplasmic and nuclear face of the NPC. This result is a major milestone in the NPC research field, solving a mystery that is elusive for decades. It opens up many questions about NPC assembly, nuclear transport and structural organization. Figure 1: Illustration Visualizing the Three-Dimensional Arrangement of the hNup107 and Its Adjacent Subcomplexes within the NPC Scaffold. The inner and outer instances are colored green and blue, respectively. Adjacent Nups and subcomplexes are shown color coded. Figure taken from Figure 7, Cell 155:1233-43.
Data: CORDIS, © European Union
Project objective
Nuclear Pore Complexes (NPCs) represent the exclusive passageway in and out of the nucleus but also function in gene regulation and nuclear organization. NPCs serve as docking sites for many viruses and were associated with several human diseases, e.g. leukemia. Understanding the nuclear transport mechanism will have a huge impact on human health research of such diseases. Multiple copies of ~30 different protein components, termed nucleoporins (Nups), form the major building blocks of NPC. Nups are organized as repetitive modules, called subcomplexes. The subcomplexes, in turn, form the scaffold of NPC in an unknown arrangement. Despite their importance, structure determination of NPCs is a considerable challenge, primarily due to their sheer size.To generate an atomic model of the human NPC, data at the different resolution levels have to be systematically collected and integrated. Determining the position of Nup subcomplexes within the NPC will be a critical step to bridge the resolution gap between different structural techniques. Since the precision of 2D-immuno-gold labeling techniques is insufficient to associate individual Nups with distinct features of the NPC structure from cryo-electron tomography (cryo-ET), alternative strategies are needed to achieve this goal. Here I propose to improve the resolution of the human NPC structure to allow assigning subcomplexes into the overall assembly. This will be achieved by cryo-ET, subtomogram averaging and further development of algorithms correcting for NPC plasticity. Furthermore, I will determine the precise positioning of subcomplexes by obtaining 3D structures of NPCs containing tagged Nups. This will contribute significantly to our understanding of the overall organization of the NPC and thus, nuclear tranport mechanism. This proposed project will give me the opportunity to learn new skills, acquire competencies and, thus pave the way towards an independent career.
Original text from CORDIS.
Participants
- EUROPEAN MOLECULAR BIOLOGY LABORATORY · HeidelbergCoordinatorGermany
Links
Data: CORDIS, © European Union