Super-resolved 3D tracking of cargo transport through nuclear pore complexes.
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ABSTRACT:
SUBMITTER: Chowdhury R
PROVIDER: S-EPMC8820391 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The nuclear pore complex (NPC) mediates all exchange between the cytoplasm and the nucleus. Small molecules can passively diffuse through the NPC, whereas larger cargos require transport receptors to translocate. How the NPC facilitates the translocation of transport receptor/cargo complexes remains unclear. To investigate this process, we tracked single protein-functionalized quantum dot cargos as they moved through human NPCs. Here we show that import proceeds by successive substeps comprising cargo capture, filtering and translocation, and release into the nucleus. Most quantum dots are rejected at one of these steps and return to the cytoplasm, including very large cargos that abort at a size-selective barrier. Cargo movement in the central channel is subdiffusive and cargos that can bind more transport receptors diffuse more freely. Without Ran GTPase, a critical regulator of transport directionality, cargos still explore the entire NPC, but have a markedly reduced probability of exit into the nucleus, suggesting that NPC entry and exit steps are not equivalent and that the pore is functionally asymmetric to importing cargos. The overall selectivity of the NPC seems to arise from the cumulative action of multiple reversible substeps and a final irreversible exit step.
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Project description:Macromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG-repeat domains in NPCs are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. We found that increasing O-GlcNAc modification of the NPC accelerated NTR-facilitated transport of proteins in both directions, and decreasing modification slowed transport. Superresolution imaging revealed strong enrichment of O-GlcNAc at the FG-repeat barrier. O-GlcNAc modification also accelerated passive permeation of a small, inert protein through NPCs. We conclude that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the nonspecific permeability of the FG-repeat barrier, perhaps by steric inhibition of interactions between FG repeats.
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Project description:Molecular transport across the nuclear envelope in eukaryotic cells is solely controlled by the nuclear pore complex (NPC). The NPC provides two types of nucleocytoplasmic transport: passive diffusion of small molecules and active chaperon-mediated translocation of large molecules. It has been shown that the interaction between intrinsically disordered proteins that line the central channel of the NPC and the transporting cargoes is the determining factor, but the exact mechanism of transport is yet unknown. Here, we use coarse-grained molecular dynamics simulations to quantify the energy barrier that has to be overcome for molecules to pass through the NPC. We focus on two aspects of transport. First, the passive transport of model cargo molecules with different sizes is studied and the size selectivity feature of the NPC is investigated. Our results show that the transport probability of cargoes is significantly reduced when they are larger than ?5 nm in diameter. Secondly, we show that incorporating hydrophobic binding spots on the surface of the cargo effectively decreases the energy barrier of the pore. Finally, a simple transport model is proposed which characterizes the energy barrier of the NPC as a function of diameter and hydrophobicity of the transporting particles.
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Project description:The nuclear pore complex (NPC) is a large protein nanopore that solely mediates molecular transport between the nucleus and cytoplasm of a eukaryotic cell. There is a long-standing consensus that selective transport barriers of the NPC are exclusively based on hydrophobic repeats of phenylalanine and glycine (FG) of nucleoporins. Herein, we reveal experimentally that charged residues of amino acids intermingled between FG repeats can modulate molecular transport through the NPC electrostatically and in a pathway-dependent manner. Specifically, we investigate the NPC of the Xenopus oocyte nucleus to find that excess positive charges of FG-rich nucleoporins slow down passive transport of a polycationic peptide, protamine, without affecting that of a polyanionic pentasaccharide, Arixtra, and small monovalent ions. Protamine transport is slower with a lower concentration of electrolytes in the transport media, where the Debye length becomes comparable to the size of water-filled spaces among the gel-like network of FG repeats. Slow protamine transport is not affected by the binding of a lectin, wheat germ agglutinin, to the peripheral route of the NPC, which is already blocked electrostatically by adjacent nucleoporins that have more cationic residues than anionic residues and even FG dipeptides. The permeability of NPCs to the probe ions is measured by scanning electrochemical microscopy using ion-selective tips based on liquid/liquid microinterfaces and is analysed by effective medium theory to determine the sizes of peripheral and central routes with distinct protamine permeability. Significantly, nanoscale electrostatic gating at the NPC can be relevant not only chemically and biologically, but also biomedically for efficient nuclear import of genetically therapeutic substances.
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Project description:A selective barrier formed by intrinsically disordered Phe-Gly (FG) nucleoporins (Nups) allows transport receptor (TR)-facilitated translocation of signal-dependent cargos through the nuclear pore complexes (NPCs) of eukaryotic cells. However, the configuration of the FG-Nup barrier and its interactions with multiple TRs in native NPCs remain obscure. Here, we mapped the interaction sites of various TRs or FG segments within the FG-Nup barrier by using high-speed super-resolution microscopy and used these sites to reconstruct the three-dimensional tomography of the native barrier in the NPC. We found that each TR possesses a unique interaction zone within the FG-Nup barrier and that two major TRs, importin β1 and Crm1, outcompete other TRs in binding FG Nups. Moreover, TRs may alter the tomography of the FG-Nup barrier and affect one another's pathways under circumstances of heavy competition.
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Project description:Exchange of macromolecules between the nucleus and cytoplasm is a key regulatory event in the expression of a cell's genome. This exchange requires a dedicated transport system: (1) nuclear pore complexes (NPCs), embedded in the nuclear envelope and composed of proteins termed nucleoporins (or "Nups"), and (2) nuclear transport factors that recognize the cargoes to be transported and ferry them across the NPCs. This transport is regulated at multiple levels, and the NPC itself also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes. Here we summarize how the yeast Saccharomyces has been used extensively as a model system to understand the fundamental and highly conserved features of this transport system, revealing the structure and function of the NPC; the NPC's role in the regulation of gene expression; and the interactions of transport factors with their cargoes, regulatory factors, and specific nucleoporins.
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Project description:Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoprotein complexes across the double-membrane nuclear envelope. In vitro studies with purified transport cofactors have revealed a general scheme of cofactor-dependent transport energetically driven by the G protein Ran. However, the size and complexity of NPCs have made it difficult to clearly define the loci and kinetics of the cofactor-NPC interactions required for transport. We now report the use of single-molecule fluorescence microscopy to directly monitor a model protein substrate undergoing transport through NPCs in permeabilized cells. This substrate, NLS-2xGFP, interacts with NPCs for an average of 10 +/- 1 ms during transport. However, because the maximum nuclear accumulation rate of NLS-2xGFP was measured to be at least approximately 10(3) molecules per NPC per s, NPCs must be capable of transporting at least approximately 10 substrate molecules simultaneously. Molecular tracking reveals that substrate molecules spend most of their transit time randomly moving in the central pore of the NPC and that the rate-limiting step is escape from the central pore.
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Project description:The cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a common histopathological hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum (ALS/FTD). However, the composition of aggregates and their contribution to the disease process remain unknown. Here we used proximity-dependent biotin identification (BioID) to interrogate the interactome of detergent-insoluble TDP-43 aggregates and found them enriched for components of the nuclear pore complex and nucleocytoplasmic transport machinery. Aggregated and disease-linked mutant TDP-43 triggered the sequestration and/or mislocalization of nucleoporins and transport factors, and interfered with nuclear protein import and RNA export in mouse primary cortical neurons, human fibroblasts and induced pluripotent stem cell-derived neurons. Nuclear pore pathology is present in brain tissue in cases of sporadic ALS and those involving genetic mutations in TARDBP and C9orf72. Our data strongly implicate TDP-43-mediated nucleocytoplasmic transport defects as a common disease mechanism in ALS/FTD.
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