Explore the vast range of titles available.

Nuclear pore complexes (NPCs) are very large proteinaceous assemblies that consist of more than 500 individual proteins1,2. NPCs are essential for nucleocytoplasmic transport of different cellular components, and disruption of the integrity of NPCs has been linked to aging, cancer and neurodegenerative diseases3–7. However, the mechanism by which membrane-embedded NPCs are turned over is currently unknown. Here we show that, after nitrogen starvation or genetic interference with the architecture of NPCs, nucleoporins are rapidly degraded in the budding yeast Saccharomyces cerevisiae. We demonstrate that NPC turnover involves vacuolar proteases and the core autophagy machinery. Autophagic degradation is mediated by the cytoplasmically exposed Nup159, which serves as intrinsic cargo receptor and directly binds to the autophagy marker protein Atg8. Autophagic degradation of NPCs is therefore inducible, enabling the removal of individual NPCs from the nuclear envelope.Lee et al. show that, after nitrogen starvation and genetic interference with the architecture of nuclear pore complexes, nucleoporins are degraded by autophagy, constituting a quality-control step at the nuclear envelope.

Cryo electron tomography with subsequent subtomogram averaging is a powerful technique to structurally analyze macromolecular complexes in their native context. Although close to atomic resolution in principle can be obtained, it is not clear how individual experimental parameters contribute to the attainable resolution. Here, we have used immature HIV-1 lattice as a benchmarking sample to optimize the attainable resolution for subtomogram averaging. We systematically tested various experimental parameters such as the order of projections, different angular increments and the use of the Volta phase plate. We find that although any of the prominently used acquisition schemes is sufficient to obtain subnanometer resolution, dose-symmetric acquisition provides considerably better outcome. We discuss our findings in order to provide guidance for data acquisition. Our data is publicly available and might be used to further develop processing routines. Here the authors systematically benchmark cryo-electron tomography acquisition schemes to optimize the attainable resolution for subtomogram averaging, and find that dose-symmetric acquisition with even angular sampling provides a better outcome than most currently used acquisition schemes.

Degradation of heparan sulfate (HS), a glycosaminoglycan (GAG) comprised of repeating units of N -acetylglucosamine and glucuronic acid, begins in the cytosol and is completed in the lysosomes. Acetylation of the terminal non-reducing amino group of α-D-glucosamine of HS is essential for its complete breakdown into monosaccharides and free sulfate. Heparan-α-glucosaminide N -acetyltransferase (HGSNAT), a resident of the lysosomal membrane, catalyzes this essential acetylation reaction by accepting and transferring the acetyl group from cytosolic acetyl-CoA to terminal α-D-glucosamine of HS in the lysosomal lumen. Mutation-induced dysfunction in HGSNAT causes abnormal accumulation of HS within the lysosomes and leads to an autosomal recessive neurodegenerative lysosomal storage disorder called mucopolysaccharidosis IIIC (MPS IIIC). There are no approved drugs or treatment strategies to cure or manage the symptoms of, MPS IIIC. Here, we use cryo-electron microscopy (cryo-EM) to determine a high-resolution structure of the HGSNAT-acetyl-CoA complex, the first step in the HGSNAT-catalyzed acetyltransferase reaction. In addition, we map the known MPS IIIC mutations onto the structure and elucidate the molecular basis for mutation-induced HGSNAT dysfunction.

The partitioning of cellular components between the nucleus and cytoplasm is the defining feature of eukaryotic life. The nuclear pore complex (NPC) selectively gates the transport of macromolecules between these compartments, but it is unknown whether surveillance mechanisms exist to reinforce this function. By leveraging in situ cryo-electron tomography to image the native cellular environment of Chlamydomonas reinhardtii, we observed that nuclear 26S proteasomes crowd around NPCs. Through a combination of subtomogram averaging and nanometer-precision localization, we identified two classes of proteasomes tethered via their Rpn9 subunits to two specific NPC locations: binding sites on the NPC basket that reflect its eightfold symmetry and more abundant binding sites at the inner nuclear membrane that encircle the NPC. These basket-tethered and membrane-tethered proteasomes, which have similar substrate-processing state frequencies as proteasomes elsewhere in the cell, are ideally positioned to regulate transcription and perform quality control of both soluble and membrane proteins transiting the NPC.

The most comprehensive architectural model to date of the nuclear pore complex reveals previously unknown local interactions, and a role for nucleoporin 358 in Y-complex oligomerization. A detailed model of the human nuclear pore complex The transport of materials between the nucleus and cytoplasm in eukaryotic cells is controlled by the nuclear pore complex. Martin Beck and colleagues have used cryo-electron tomography, mass spectrometry and other analyses to generate the most comprehensive architectural model of the human nuclear pore complex to date. The model reveals previously unknown local interactions, and a role for the transport channel nucleoporin 358 (Nup358) in mediating oligomerization of the Y-complex within the nuclear pore complex. Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter 1 . The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ . Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block—although compositionally identical—engage in different local sets of interactions and conformations.

Nuclear pore complexes (NPCs) are 110-megadalton assemblies that mediate nucleocytoplasmic transport. NPCs are built from multiple copies of ~30 different nucleoporins, and understanding how these nucleoporins assemble into the NPC scaffold imposes a formidable challenge. Recently, it has been shown how the Y complex, a prominent NPC module, forms the outer rings of the nuclear pore. However, the organization of the inner ring has remained unknown until now. We used molecular modeling combined with cross-linking mass spectrometry and cryo-electron tomography to obtain a composite structure of the inner ring. This architectural map explains the vast majority of the electron density of the scaffold. We conclude that despite obvious differences in morphology and composition, the higher-order structure of the inner and outer rings is unexpectedly similar.

Nuclear pore complexes (NPCs) span the nuclear envelope and mediate nucleocytoplasmic exchange. They are a hallmark of eukaryotes and deeply rooted in the evolutionary origin of cellular compartmentalization. NPCs have an elaborate architecture that has been well studied in vertebrates. Whether this architecture is unique or varies significantly in other eukaryotic kingdoms remains unknown, predominantly due to missing in situ structural data. Here, we report the architecture of the algal NPC from the early branching eukaryote Chlamydomonas reinhardtii and compare it to the human NPC. We find that the inner ring of the Chlamydomonas NPC has an unexpectedly large diameter, and the outer rings exhibit an asymmetric oligomeric state that has not been observed or predicted previously. Our study provides evidence that the NPC is subject to substantial structural variation between species. The divergent and conserved features of NPC architecture provide insights into the evolution of the nucleocytoplasmic transport machinery. While the architecture of vertebrate nuclear pore complexes (NPCs) is well understood, the extent of its evolutionary conservation is still unclear. Here, the authors analyze the in situ architecture of an algal NPC, revealing distinct structural features that provide insights into NPC evolution.

Klar et al. explore the structure and polarity of biogenic hemozoin crystals, historically known as the malaria pigment.

The Tandem of pore domain in a Weak Inward Rectifying K + channel 2 (TWIK-2; KCNK6 ) is a member of the Two-Pore Domain K + (K 2P ) channel family, which is associated with pulmonary hypertension, lung injury, and inflammation. Despite its physiological relevance, the structure, regulatory mechanisms, and selective modulators of TWIK-2 remain largely unknown. Here, we present a 3.7 Å single particle cryo-electron microscopy structure of human TWIK-2 and highlight its conserved and distinctive features. Using automated whole-cell patch clamp recordings, we demonstrate that gating in TWIK-2 is voltage-dependent and insensitive to changes in the extracellular pH. We identify key residues that influence TWIK-2 activity by employing site-directed mutagenesis and provide insights into the possible lipid-mediated mechanism of TWIK-2 regulation. Additionally, we demonstrate the application of high-throughput automated whole-cell patch clamp platforms to screen small molecule modulators of TWIK-2. Our work serves as a foundation for designing high-throughput small molecule screening campaigns to identify specific high-affinity TWIK-2 modulators, including promising- anti-inflammatory therapeutics. TWIK-2 is an endolysosomal potassium channel implicated in inflammatory responses. Here, authors present a cryo-EM structure of human TWIK-2 and establish a high-throughput automated patch-clamp electrophysiology assay to investigate modulation of TWIK-2.

Rapid structural analysis of purified proteins and their complexes has become increasingly common thanks to key methodological advances in cryo-electron microscopy (cryo-EM) and associated data processing software packages. In contrast, analogous structural analysis in cells via cryo-electron tomography (cryo-ET) remains challenging due to critical technical bottlenecks, including low-throughput sample preparation and imaging, and laborious data processing methods. Here, we describe a rapid in situ cryo-ET sample preparation and data analysis workflow that results in the routine determination of sub-nm resolution ribosomal structures. We apply this workflow to E. coli , producing a 5.8 Å structure of the 70S ribosome from cells in less than 10 days and facilitating the discovery of a minor population of 100S-like disomes. We envision our approach to be widely applicable to related bacterial samples. A workflow for rapid cryo-ET structural characterization of bacterial ribosomes in situ and the identification of a 100S-like disome structure.