Dr. Martin Beck from the European Molecular Biology Laboratory in Heidelberg was awarded an ERC Starting Grant for his project “Atlas of Cell-Type Specific Nuclear Pore Complex Structures”. The combination of cryo-electron microscope tomography, proteomics and biochemical methods will enable the creation of a high-resolution three-dimensional image of the nuclear pore complex.
The double membrane, which separates the chromosome-containing nuclear space (karyoplasm or nucleoplasm) from the cytoplasm, is a fundamental characteristic of eukaryotic cells. In the majority of cells, the nuclear membrane is connected to the endoplasmic reticulum, but differs from the latter and all other membrane systems due to the presence of nuclear pores that enable the controlled exchange of molecules (in particular RNAs and proteins) between the nucleo- and the cytoplasm.
Since Werner Franke (then at the University of Freiburg, later at the German Cancer Research Center) carried out electron microscope investigations of isolated nuclear membranes in the late-1960s, it has been known that the nuclear pores are not just simple holes in the membrane, but complex three-dimensional structures consisting of numerous components. Many researchers over the last twenty years, including the Nobel Laureate Günter Blobel and the biochemist Ed Hurt from Heidelberg (see BIOPRO article entitled "In vitro modeling of the nuclear pore complex of a thermophilic fungus"), have studied the structure and function of nuclear pores in great detail. It is now known that the nuclear pore complex is not just one of the biggest supramolecular cell structures consisting of at least 30 different proteins (nucleoporins), but that it is also a dynamic machine associated with many other components that enable the regulated and controlled transport of macromolecules between the nuclear space and the cytoplasm.
It is also assumed that the nuclear pore complex plays a role in the segregation of chromosomes prior to cell division (mitosis). While the organisation of the nuclear pore complex seems to have remained highly conserved throughout evolution, some of the species investigated display characteristic differences in terms of composition and function. In higher organisms, the nuclear pore complex (NPC) disintegrates during mitosis and the dissolution of the nuclear membrane and reassembles in the daughter cells after cell division. In many lower eukaryotes such as yeasts, for example, the nuclear membrane and the nuclear pores remain intact when the cells divide.
Dr. Martin Beck, head of a group of researchers at the European Molecular Biology Laboratory in Heidelberg, has made a name for himself with his achievements in elucidating the structure and function of molecular machines such as NPCs. He used innovative proteomics methods and cryo-electron microscopy for his examinations. In September 2012, the European Research Council (ERC) announced that it had selected Beck as one of the young European scientists who would receive a renowned ERC Starting Grant. The ERC gave Beck the grant for a project aimed at developing an atlas of the cell-type specific structures of the nuclear pore complex.
Beck did his doctoral thesis in the Molecular Structural Biology department led by Professor Dr. Wolfgang Baumeister at the Max Planck Institute of Biochemistry in Martinsried, and at this stage he was already focusing on the structure of nuclear pores. Back then, Baumeister and his team were, and still are, worldwide leaders in the development of cryo-electron tomography (CET), a unique technology that allows the three-dimensional representation of macromolecular complexes in their native environment. A series of electron microscope images of snap-frozen cells and cell components is produced in a similar way to computed tomography and assembled into a 3D image by a computer. The sample can be rotated in order to obtain images from different angles. The procedure has the potential to close the gap between the molecular and cellular dimensions in the field of structural biology. Using statistical analyses of around 250 NPCs recorded from different angles, Beck and his colleagues were able to reconstruct the three-dimensional structure of the pore complex and assign it to different functional states. They have shown that the structures of high electron density of the pore channels are not integral NPC components; instead they have been found to be cargo molecules, i.e. particles that are transported around.
After completing his doctorate, Martin Beck joined the laboratory of Professor Ruedi Aebersold at the Swiss Federal Institute of Technology in Zurich in 2006. Ruedi Aebersold is a pioneer of proteome research. New developments in the field of proteomics excellently complement CET investigations in the acquisition of information about the function of NCPs and the development of high-resolution models of these molecular machines. Mass spectrometry can be used to investigate complex protein mixtures and to obtain quantitative information about the proportions of the protein components in the complexes. This usually involves using specifically labelled reference peptides. The spatial orientation of proteins can be determined by carefully linking the individual components with each other. Beck has been the head of the research group “Structure and Function of Large Macromolecular Assemblies” at the European Molecular Biology Laboratory in Heidelberg since 2010. The team not only investigates NPCs, but also other macromolecular complexes consisting of many different components, including proteasomes. In order to do this, the team use CET and proteomics methods which Beck first learned to use in the laboratories of Baumeister and Aebersold and which he has developed further.
In Martinsried, Beck investigated the nuclear pores of nuclei isolated from Dictyostelium discoideum, a fungus Professor Günther Gerisch helped to make the model organsim in the field of cell biology. Information about the NPCs of a number of organisms of different evolutionary stages is now available, including unicellular yeasts, clawed frogs (Xenopus) and humans. The structure of human NPCs is very similar to that of Xenopus NPCs whereas the NPCs of lower eukaryotes like Dictyostelium differ considerably. Characteristic differences have also been found between certain cell types with different functions within a single species. The ERC Starting Grant will now enable Beck and his team to systematically investigate cell-type specific NPC structures. The researchers hope that they will be able to represent the structures in action and gain new insights into how the selective energy-dependent transport of large macromolecules through the pore channel works. In addition, they also hope to elucidate the processes that enable the individual components of the nuclear pore complex to assemble into a ‘gigantic’ machinery with a molecular weight of more than 100 million Da and disintegrate again when the cell divides and the nuclear membrane dissolves.