Visualizing cellular processes at the molecular level by cryo-electron tomography.

The cellular landscape rapidly changes throughout the biological processes that transpire within a cell. For example, the cytoskeleton is remodeled within fractions of a second. Therefore, reliable structural analysis of the cell requires approaches that allow for instantaneous arrest of functional states of a given process while offering the best possible preservation of the delicate cellular structure. Electron tomography of vitrified but otherwise unaltered cells (cryo-ET) has proven to be the method of choice for three-dimensional (3D) reconstruction of cellular architecture at a resolution of 4-6 nm. Through the use of cryo-ET, the 3D organization of macromolecular complexes and organelles can be studied in their native environment in the cell. In this Commentary, we focus on the application of cryo-ET to study eukaryotic cells - in particular, the cytoskeletal-driven processes that are involved in cell movements, filopodia protrusion and viral entry. Finally, we demonstrate the potential of cryo-ET to determine structures of macromolecular complexes in situ, such as the nuclear pore complex.

The human nuclear pore complex as revealed by cryo-electron tomography.

Nuclear pore complexes (NPCs) are the sole passage through the nuclear envelope, connecting the cytoplasm to the nucleoplasm. These gigantic molecular machines, over 100 MDa in molecular weight, allow free diffusion of small molecules and ions while mediating selective energy-dependent nucleocytoplasmic transport of large macromolecules. Here, we applied cryo-electron tomography to human fibroblast cells, reconstructing their nuclear envelopes without applying any purification steps. From these reconstructions, we extracted subtomograms containing individual NPCs and utilized in silico subtomogram averaging procedures to determine the structure of the mammalian pore complex at a resolution of ∼6.6 nm. Beyond revealing the canonical features of the human NPC, our analysis identified inner lateral channels and fusing bridge-like structures, suggesting alternative routes of peripheral nuclear passage. Finally, we concluded from our structural analysis that the human NPC is structurally distinct from that of lower eukaryotes in terms of dimension and organization but resembles its amphibian (frog) counterpart.

Cryo-electron tomography: gaining insight into cellular processes by structural approaches.

Visualization of cellular processes at a resolution of the individual protein should involve integrative and complementary approaches that can eventually draw realistic functional and cellular landscapes. Electron tomography of vitrified but otherwise unaltered cells emerges as a central method for three-dimensional reconstruction of cellular architecture at a resolution of 2-6 nm. While a combination of correlative light-based microscopy with cryo-electron tomography (cryo-ET) provides medium-resolution insight into pivotal cellular processes, fitting high-resolution structural approaches, for example, X-ray crystallography, into reconstructed macromolecular assemblies provides unprecedented information on native protein assemblies. Thus, cryo-ET bridges the resolution gap between cellular and structural biology. In this article, we focus on the study of eukaryotic cells and macromolecular complexes in a close-to-life-state. We discuss recent developments and structural findings enabling major strides to be made in understanding complex physiological functions.

Perspective on the metazoan nuclear pore complex.

Fusing the inner and outer membranes of the nucleus, the nuclear pore complex (NPC) forms a selective portal which serves as the sole gateway of the nucleus. These aqueous translocation channels allow free diffusion of small molecules and ions, as well as receptor-mediated transport of large macromolecules. Over the last several years major progress has been made in both structural determination of individual nucleopurins (Nups) and their complexes by X-ray crystallography and in structural analysis of the entire assembly by means of cryo-electron tomography. By combining cryo-electron tomography with advanced image processing techniques, the metazoan NPC structure from Xenopus oocytes was resolved to medium resolution, revealing novel details. Here, we discuss new features of the Xenopus NPC and consider future perspectives that will eventually allow resolution of the structure and function of NPCs with high accuracy.

Structural analysis of the nuclear pore complex by integrated approaches.

In eukaryotic cells, the nucleus is surrounded by a double membrane system, the nuclear envelope (NE), in which the outer membrane is continuous with the endoplasmic reticulum (ER). Nuclear pore complexes (NPCs) fuse the inner and outer nuclear membranes to form aqueous translocation channels that allow the free diffusion of small molecules and ions, as well as receptor-mediated transport of large macromolecules. Being the sole gateways for import and export to and from the nucleus, NPCs regulate the nucleocytoplasmic transport of macromolecules in a highly selective manner to maintain cellular functions. The large size and complexity of these multimolecular assemblies, which are composed of approximately 30 different proteins (termed nucleoporins), present a major challenge for structural biologists. Here, we discuss the latest structural findings related to the functional organization of the NPC.