Structural investigation of eukaryotic cells: From the periphery to the interior by cryo-electron tomography

Cryo-electron tomography (cryo-ET) combines a close-to-life preservation of the cell with high-resolution three-dimensional (3D) imaging. This allows to study the molecular architecture of the cellular landscape and provides unprecedented views on biological processes and structures. In this review we mainly focus on the application of cryo-ET to visualize and structurally characterize eukaryotic cells - from the periphery to the cellular interior. We discuss strategies that can be employed to investigate the structure of challenging targets in their cellular environment as well as the application of complimentary approaches in conjunction with cryo-ET

The Nuclear Pore Complex: Birth, Life, and Death of a Cellular Behemoth

Nuclear pore complexes (NPCs) are the only transport channels that cross the nuclear envelope. Constructed from ~500–1000 nucleoporin proteins each, they are among the largest macromolecular assemblies in eukaryotic cells. Thanks to advances in structural analysis approaches, the construction principles and architecture of the NPC have recently been revealed at submolecular resolution. Although the overall structure and inventory of nucleoporins are conserved, NPCs exhibit significant compositional and functional plasticity even within single cells and surprising variability in their assembly pathways. Once assembled, NPCs remain seemingly unexchangeable in post-mitotic cells. There are a number of as yet unresolved questions about how the versatility of NPC assembly and composition is established, how cells monitor the functional state of NPCs or how they could be renewed. Here, we review current progress in our understanding of the key aspects of NPC architecture and lifecycle.publishedVersio

An amphipathic helix in Brl1 is required for nuclear pore complex biogenesis in S. cerevisiae

The nuclear pore complex (NPC) is the central portal for macromolecular exchange between the nucleus and cytoplasm. In all eukaryotes, NPCs assemble into an intact nuclear envelope (NE) during interphase, but the process of NPC biogenesis remains poorly characterized. Furthermore, little is known about how NPC assembly leads to the fusion of the outer and inner NE, and no factors have been identified that could trigger this event. Here, we characterize the transmembrane protein Brl1 as an NPC assembly factor required for NE fusion in budding yeast. Brl1 preferentially associates with NPC assembly intermediates and its depletion halts NPC biogenesis, leading to NE herniations that contain inner and outer ring nucleoporins but lack the cytoplasmic export platform. Furthermore, we identify an essential amphipathic helix in the luminal domain of Brl1 that mediates interactions with lipid bilayers. Mutations in this amphipathic helix lead to NPC assembly defects, and cryo-electron tomography analyses reveal multilayered herniations of the inner nuclear membrane with NPC-like structures at the neck, indicating a failure in NE fusion. Taken together, our results identify a role for Brl1 in NPC assembly and suggest a function of its amphipathic helix in mediating the fusion of the inner and outer nuclear membranes

An amphipathic helix in Brl1 is required for nuclear pore complex biogenesis in S. cerevisiae

The nuclear pore complex (NPC) is the central portal for macromolecular exchange between the nucleus and cytoplasm. In all eukaryotes, NPCs assemble into an intact nuclear envelope (NE) during interphase, but the process of NPC biogenesis remains poorly characterized. Furthermore, little is known about how NPC assembly leads to the fusion of the outer and inner NE, and no factors have been identified that could trigger this event. Here, we characterize the transmembrane protein Brl1 as an NPC assembly factor required for NE fusion in budding yeast. Brl1 preferentially associates with NPC assembly intermediates and its depletion halts NPC biogenesis, leading to NE herniations that contain inner and outer ring nucleoporins but lack the cytoplasmic export platform. Furthermore, we identify an essential amphipathic helix in the luminal domain of Brl1 that mediates interactions with lipid bilayers. Mutations in this amphipathic helix lead to NPC assembly defects, and cryo-electron tomography analyses reveal multilayered herniations of the inner nuclear membrane with NPC-like structures at the neck, indicating a failure in NE fusion. Taken together, our results identify a role for Brl1 in NPC assembly and suggest a function of its amphipathic helix in mediating the fusion of the inner and outer nuclear membranes.publishedVersio

Cellular and Structural Studies of Eukaryotic Cells by Cryo-Electron Tomography

The architecture of protein assemblies and their remodeling during physiological processes is fundamental to cells. Therefore, providing high-resolution snapshots of macromolecular complexes in their native environment is of major importance for understanding the molecular biology of the cell. Cellular structural biology by means of cryo-electron tomography (cryo-ET) offers unique insights into cellular processes at an unprecedented resolution. Recent technological advances have enabled the detection of single impinging electrons and improved the contrast of electron microscopic imaging, thereby significantly increasing the sensitivity and resolution. Moreover, various sample preparation approaches have paved the way to observe every part of a eukaryotic cell, and even multicellular specimens, under the electron beam. Imaging of macromolecular machineries at high resolution directly within their native environment is thereby becoming reality. In this review, we discuss several sample preparation and labeling techniques that allow the visualization and identification of macromolecular assemblies in situ, and demonstrate how these methods have been used to study eukaryotic cellular landscapes

Cellular and Structural Studies of Eukaryotic Cells by Cryo-Electron Tomography

The architecture of protein assemblies and their remodeling during physiological processes is fundamental to cells. Therefore, providing high-resolution snapshots of macromolecular complexes in their native environment is of major importance for understanding the molecular biology of the cell. Cellular structural biology by means of cryo-electron tomography (cryo-ET) offers unique insights into cellular processes at an unprecedented resolution. Recent technological advances have enabled the detection of single impinging electrons and improved the contrast of electron microscopic imaging, thereby significantly increasing the sensitivity and resolution. Moreover, various sample preparation approaches have paved the way to observe every part of a eukaryotic cell, and even multicellular specimens, under the electron beam. Imaging of macromolecular machineries at high resolution directly within their native environment is thereby becoming reality. In this review, we discuss several sample preparation and labeling techniques that allow the visualization and identification of macromolecular assemblies in situ, and demonstrate how these methods have been used to study eukaryotic cellular landscapes

Cellular and Structural Studies of Eukaryotic Cells by Cryo-Electron Tomography

The architecture of protein assemblies and their remodeling during physiological processes is fundamental to cells. Therefore, providing high-resolution snapshots of macromolecular complexes in their native environment is of major importance for understanding the molecular biology of the cell. Cellular structural biology by means of cryo-electron tomography (cryo-ET) offers unique insights into cellular processes at an unprecedented resolution. Recent technological advances have enabled the detection of single impinging electrons and improved the contrast of electron microscopic imaging, thereby significantly increasing the sensitivity and resolution. Moreover, various sample preparation approaches have paved the way to observe every part of a eukaryotic cell, and even multicellular specimens, under the electron beam. Imaging of macromolecular machineries at high resolution directly within their native environment is thereby becoming reality. In this review, we discuss several sample preparation and labeling techniques that allow the visualization and identification of macromolecular assemblies in situ, and demonstrate how these methods have been used to study eukaryotic cellular landscapes.ISSN:2073-440

The Nuclear Pore Complex: Birth, Life, and Death of a Cellular Behemoth

Nuclear pore complexes (NPCs) are the only transport channels that cross the nuclear envelope. Constructed from ~500–1000 nucleoporin proteins each, they are among the largest macromolecular assemblies in eukaryotic cells. Thanks to advances in structural analysis approaches, the construction principles and architecture of the NPC have recently been revealed at submolecular resolution. Although the overall structure and inventory of nucleoporins are conserved, NPCs exhibit significant compositional and functional plasticity even within single cells and surprising variability in their assembly pathways. Once assembled, NPCs remain seemingly unexchangeable in post-mitotic cells. There are a number of as yet unresolved questions about how the versatility of NPC assembly and composition is established, how cells monitor the functional state of NPCs or how they could be renewed. Here, we review current progress in our understanding of the key aspects of NPC architecture and lifecycle.ISSN:2073-440

The Nuclear Pore Complex: Birth, Life, and Death of a Cellular Behemoth

Nuclear pore complexes (NPCs) are the only transport channels that cross the nuclear envelope. Constructed from ~500–1000 nucleoporin proteins each, they are among the largest macromolecular assemblies in eukaryotic cells. Thanks to advances in structural analysis approaches, the construction principles and architecture of the NPC have recently been revealed at submolecular resolution. Although the overall structure and inventory of nucleoporins are conserved, NPCs exhibit significant compositional and functional plasticity even within single cells and surprising variability in their assembly pathways. Once assembled, NPCs remain seemingly unexchangeable in post-mitotic cells. There are a number of as yet unresolved questions about how the versatility of NPC assembly and composition is established, how cells monitor the functional state of NPCs or how they could be renewed. Here, we review current progress in our understanding of the key aspects of NPC architecture and lifecycle

Mapping the native organization of the yeast nuclear pore complex using nuclear radial intensity measurements

ISSN:0027-8424ISSN:1091-649