CRYO ET SYNAPSE · Three dimensional organization of dendritic spines : cryo electron tomography studies

Final Activity Report Summary - CRYO ET SYNAPSE (three dimensional organisation of dendritic spines : Cryo electron tomography studies)

During the course of my Marie Curie fellowship the researcher expertised in the lab headed by Prof. Wolfgang Baumeister, a leading group in the field of three dimensional electron microscopy (3DEM) and in particular cryo electron tomography (cryo ET). Cryo ET emerged as a powerful tool for the study of macromolecular assemblies preserved in their native cellular environment. The main goal of this fellowship was to acquire the necessary methodologies required to address fundamental biological questions using cryo ET as a main tool. The study presented here described the determination of the 3D structure of the nuclear pore complex (NPC), the largest and presumably most complex macromolecular machine in the cell, using cryo ET as the main research tool. The genetic material, the atomic blueprint of the organism, is the most important component in the living cell. In eukaryotic cells it is condensed within the nucleus where it is segregated from the cytoplasm. While the nucleus requires this protective isolation, communication with the rest of the cell is essential. Whereas transcription of genes to mRNA takes place in the nucleus, their translation to protein product is carried out by the ribosomes residing on the endoplasmic reticulum (ER) in the cytoplasm. On the other hand, DNA translation and transcription requires specific proteins that need to be transported from the cytoplasm into the nucleus. The nuclear pore complex (NPC) mediates the exchange of components between the nucleus and cytoplasm in a controlled manner. In order to achieve a deeper insight into the transport mechanism through the NPC, structural investigation is required. To date, high resolution structural studies of the entire NPC were hampered due to its complex composition and its dynamic nature. Cryo electron tomography is currently the method of choice for investigating the 3D structure of the NPC as it is preserved in a close-to-native state where it is embedded within the double nuclear membrane without chemical fixation or detergent extraction. Here we show the 3D structure revealed by cryo electron tomography of the NPC as resolved from Xenopus oocytes spread nuclear envelopes. In order to analyse the 3D structure of the NPC, subtomographic volumes, each containing one complete NPC structure, were excised to generate a dataset of NPC 3D particles. These particles were then aligned and averaged relative to each other implementing also the characteristic 8-fold symmetry around the axis of transport. The resulting model, extending to 10 nm resolution, shows the overall architecture of the NPC. A limiting factor in attaining higher resolution structure of the NPC is the dynamic movements of the 8 protomers composing the NPC, thus perturbing the octagonal symmetry. In a study recently published Beck and co-workers established a method to account, at least for some extent, for such movements. According to this method, the NPC is not processed as an intact particle but rather as 8 independent protomeric volumes, thus making the dataset 8 times larger. Using symmetry independent protomer averaging algorithms, the 3D subtomographic volumes were aligned and averaged, to generate a model at 6.4 nm resolution. The protomeric structures can be computationally recombined based on the 8-fold symmetry of the NPC to generate a new model for the NPC. This approach provided a substantial improvement to the structural understanding of the NPC by resolving novel canonical structures, particularly at the central spoke ring complex and lumenal connections to the membranes.

Data: CORDIS, © European Union