Super-resolved 3D Tracking of Cargo Transport Through Nuclear Pore Complexes by Astigmatism Imaging

This protocol describes a two-color astigmatic imaging approach that enables direct 3D visualization of cargo transport trajectories relative to a super-resolved octagonal double-ring scaffold structure of the nuclear pore complex (NPC). Though astigmatism imaging is commonly achieved via a cylindrical lens, this protocol utilizes an adaptive optics (AO) system, which enables optimization of the astigmatism for the precision needs of the experiment as well as correction of the focal mismatch arising from chromatic aberrations in multi-color applications. With this approach, single particle spatial precision values in x, y, and z are typically 5-20 nm, and these depend on astigmatism, photon level and position in z. The method enables resolution of transport conduits through the ~60 nm diameter pore of NPCs by particle tracking on the millisecond timescale. The success of this approach is enabled by the high rigidity of fully active NPCs within the nuclear envelope of permeabilized cells. For a detailed application of this protocol, please refer to https://www.nature.com/articles/s41556-021-00815-6. The figure and table numbers in this protocol that are indicated with an “NCB” prefix (e.g., NCB Figure X) refer to the figures and table in this reference paper.

Autophagy of the Nucleus in Health and Disease

Nucleophagy is an organelle-selective subtype of autophagy that targets nuclear material for degradation. The macroautophagic delivery of micronuclei to the vacuole, together with the nucleus-vacuole junction-dependent microautophagic degradation of nuclear material, were first observed in yeast. Nuclear pore complexes and ribosomal DNA are typically excluded during conventional macronucleophagy and micronucleophagy, indicating that degradation of nuclear cargo is tightly regulated. In mammals, similarly to other autophagy subtypes, nucleophagy is crucial for cellular differentiation and development, in addition to enabling cells to respond to various nuclear insults and cell cycle perturbations. A common denominator of all nucleophagic processes characterized in diverse organisms is the dependence on the core autophagic machinery. Here, we survey recent studies investigating the autophagic processing of nuclear components. We discuss nucleophagic events in the context of pathology, such as neurodegeneration, cancer, DNA damage, and ageing.

Transport receptor occupancy in Nuclear Pore Complex mimics

Nuclear Pore Complexes (NPCs) regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells. Intrinsically disordered Phe-Gly nucleoporins (FG Nups) line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor (karyopherin; Kap). Here, we assess 'Kap-centric' NPC models, which postulate that Kaps participate in establishing the selective barrier. We combine biomimetic nanopores, formed by tethering Nsp1 to the inner wall of a solid-state nanopore, with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores: one population that is transported across the pore in milliseconds, and a second population that is stably assembled within the FG mesh of the pore. Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh. Modeling reveals an accumulation of Kap95 near the pore wall, yielding a conductance decrease. We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that, even at high concentrations, Kaps only bind at most 8% of the FG-motifs in the nanopore, indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs. Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps, where a stable population of Kaps binds avidly to the NPC periphery, while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1.

Dunking into the Lipid Bilayer: How Direct Membrane Binding of Nucleoporins Can Contribute to Nuclear Pore Complex Structure and Assembly

Nuclear pore complexes (NPCs) mediate the selective and highly efficient transport between the cytoplasm and the nucleus. They are embedded in the two membrane structure of the nuclear envelope at sites where these two membranes are fused to pores. A few transmembrane proteins are an integral part of NPCs and thought to anchor these complexes in the nuclear envelope. In addition, a number of nucleoporins without membrane spanning domains interact with the pore membrane. Here we review our current knowledge of how these proteins interact with the membrane and how this interaction can contribute to NPC assembly, stability and function as well as shaping of the pore membrane.

Percolation transition determines protein size limit for passive transport through the nuclear pore complex

Nuclear pore complexes (NPCs) control biomolecular transport in and out of the nucleus. Disordered nucleoporins in the complex's central pore form a permeation barrier, preventing unassisted transport of large biomolecules. Here, we combine coarse-grained simulations of an experimentally-derived NPC structure with a theoretical model to determine the microscopic mechanism of passive transport. Brute-force simulations of protein diffusion through the NPC reveal telegraph-like behavior, where prolonged diffusion on one side of the NPC is interrupted by rapid crossings to the other. We rationalize this behavior using a theoretical model that reproduces the energetics and kinetics of permeation solely from statistical analysis of transient voids within the disordered mesh. As the protein size increases, the mesh transforms from a soft to a hard barrier, enabling orders-of-magnitude reduction in permeation rate for proteins beyond the percolation size threshold. Our model enables exploration of alternative NPC architectures and sets the stage for uncovering molecular mechanisms of facilitated nuclear transport.

SUMO-Based Regulation of Nuclear Positioning to Spatially Regulate Homologous Recombination Activities at Replication Stress Sites

DNA lesions have properties that allow them to escape their nuclear compartment to achieve DNA repair in another one. Recent studies uncovered that the replication fork, when its progression is impaired, exhibits increased mobility when changing nuclear positioning and anchors to nuclear pore complexes, where specific types of homologous recombination pathways take place. In yeast models, increasing evidence points out that nuclear positioning is regulated by small ubiquitin-like modifier (SUMO) metabolism, which is pivotal to maintaining genome integrity at sites of replication stress. Here, we review how SUMO-based pathways are instrumental to spatially segregate the subsequent steps of homologous recombination during replication fork restart. In particular, we discussed how routing towards nuclear pore complex anchorage allows distinct homologous recombination pathways to take place at halted replication forks.

Fragile X–Related Protein 1 Regulates Nucleoporin Localization in a Cell Cycle–Dependent Manner

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope (NE) where they ensure the transport of macromolecules between the nucleus and the cytoplasm. NPCs are built from nucleoporins (Nups) through a sequential assembly order taking place at two different stages during the cell cycle of mammalian cells: at the end of mitosis and during interphase. In addition, fragile X–related proteins (FXRPs) can interact with several cytoplasmic Nups and facilitate their localization to the NE during interphase likely through a microtubule-dependent mechanism. In the absence of FXRPs or microtubule-based transport, Nups aberrantly localize to the cytoplasm forming the so-called cytoplasmic nucleoporin granules (CNGs), compromising NPCs’ function on protein export. However, it remains unknown if Nup synthesis or degradation mechanisms are linked to the FXRP–Nup pathway and if and how the action of FXRPs on Nups is coordinated with the cell cycle progression. Here, we show that Nup localization defects observed in the absence of FXR1 are independent of active protein translation. CNGs are cleared in an autophagy- and proteasome-independent manner, and their presence is restricted to the early G1 phase of the cell cycle. Our results thus suggest that a pool of cytoplasmic Nups exists that contributes to the NPC assembly specifically during early G1 to ensure NPC homeostasis at a short transition from mitosis to the onset of interphase.

Loss of Nup210 results in muscle repair delays and age-associated alterations in muscle integrity

Nuclear pore complexes, the channels connecting the nucleus with the cytoplasm, are built by multiple copies of ∼30 proteins called nucleoporins. Recent evidence has exposed that nucleoporins can play cell type-specific functions. Despite novel discoveries into the cellular functions of nucleoporins, their role in the regulation of mammalian tissue physiology remains mostly unexplored because of a limited number of nucleoporin mouse models. Here we show that ablation of Nup210/Gp210, a nucleoporin previously identified to play a role in myoblast differentiation and Zebrafish muscle maturation, is dispensable for skeletal muscle formation and growth in mice. We found that although primary satellite cells from Nup210 knockout mice can differentiate, these animals show delayed muscle repair after injury. Moreover, Nup210 knockout mice display an increased percentage of centrally nucleated fibers and abnormal fiber type distribution as they age. Muscle function experiments also exposed that Nup210 is required for muscle endurance during voluntary running. Our findings indicate that in mammals, Nup210 is important for the maintenance of skeletal muscle integrity and for proper muscle function providing novel insights into the in vivo roles of nuclear pore complex components.