Interaction between NTF2 and xFxFG-containing nucleoporins is required to mediate nuclear import of RanGDP.

Nuclear transport factor 2 (NTF2) is a small, homodimeric protein that binds to both RanGDP and xFxFG repeat-containing nucleoporins, such as yeast Nsp1p and vertebrate p62. NTF2 is required for efficient nuclear protein import and has been shown to mediate the nuclear import of RanGDP. We have used the crystal structures of rat NTF2 and its complex with RanGDP to design a mutant, W7A-NTF2, in which the affinity for xFxFG-repeat nucleoporins is reduced while wild-type binding to RanGDP is retained. The 2.5 Å resolution crystal structure of W7A-NTF2 is virtually superimposable upon the wild-type protein structure, indicating that the mutation had not introduced a more general conformational change. Therefore, our data suggest that the exposed side-chain of residue 7 is crucial to the interaction between NTF2 and xFxFG repeat-containing nucleoporins. Consistent with its reduced affinity for xFxFG nucleoporins, fluorescently labelled W7A-NTF2 binds less strongly to the nuclear envelope of permeabilized cultured cells than wild-type NTF2 and, when microinjected into Xenopus oocytes, colloidal gold coated with W7A-NTF2 binds less strongly to the central channel of nuclear pore complexes than wild-type NTF2-coated gold. Significantly, W7A-NTF2 only weakly stimulated the nuclear import of fluorescein-labelled RanGDP, providing direct evidence that an interaction between NTF2 and xFxFG repeat-containing nucleoporins is required to mediate the nuclear import of RanGDP

Probing the potential of mucus permeability to signify preterm birth risk

Preterm birth is the leading cause of neonatal mortality, and is frequently associated with intra-amniotic infection hypothesized to arise from bacterial ascension across a dysfunctional cervical mucus plug. To study this dysfunction, we assessed the permeability of cervical mucus from non-pregnant ovulating (n = 20) and high-(n = 9) and low-risk (n = 16) pregnant women to probes of varying sizes and surface chemistries. We found that the motion of negatively charged, carboxylated microspheres in mucus from pregnant patients was significantly restricted compared to ovulating patients, but not significantly different between high-A nd low-risk pregnant women. In contrast, charged peptide probes small enough to avoid steric interactions, but sensitive to the biochemical modifications of mucus components exhibited significantly different transport profiles through mucus from high-A nd low-risk patients. Thus, although both microstructural rearrangements of the components of mucus as well as biochemical modifications to their adhesiveness may alter the overall permeability of the cervical mucus plug, our findings suggest that the latter mechanism plays a dominant role in the impairment of the function of this barrier during preterm birth. We expect that these probes may be readily adapted to study the mechanisms underlying disease progression on all mucosal epithelia, including those in the mouth, lungs, and gut.National Science Foundation (U.S.) (Award DMR-0819762)National Science Foundation (U.S.) (Award 1122374

Crystallographic and Biochemical Analysis of the Ran-Binding Zinc Finger Domain

The nuclear pore complex (NPC) resides in circular openings within the nuclear envelope and serves as the sole conduit to facilitate nucleocytoplasmic transport in eukaryotes. The asymmetric distribution of the small G protein Ran across the nuclear envelope regulates directionality of protein transport. Ran interacts with the NPC of metazoa via two asymmetrically localized components, Nup153 at the nuclear face and Nup358 at the cytoplasmic face. Both nucleoporins contain a stretch of distinct, Ran-binding zinc finger domains. Here, we present six crystal structures of Nup153-zinc fingers in complex with Ran and a 1.48 Å crystal structure of RanGDP. Crystal engineering allowed us to obtain well diffracting crystals so that all ZnF–Ran complex structures are refined to high resolution. Each of the four zinc finger modules of Nup153 binds one Ran molecule in apparently non-allosteric fashion. The affinity is measurably higher for RanGDP than for RanGTP and varies modestly between the individual zinc fingers. By microcalorimetric and mutational analysis, we determined that one specific hydrogen bond accounts for most of the differences in the binding affinity of individual zinc fingers. Genomic analysis reveals that only in animals do NPCs contain Ran-binding zinc fingers. We speculate that these organisms evolved a mechanism to maintain a high local concentration of Ran at the vicinity of the NPC, using this zinc finger domain as a sink

The Crossroads of Glycoscience, Infection, and Immunology

Advances in experimental capabilities in the glycosciences offer expanding opportunities for discovery in the broad areas of immunology and microbiology. These two disciplines overlap when microbial infection stimulates host immune responses and glycan structures are central in the processes that occur during all such encounters. Microbial glycans mediate host-pathogen interactions by acting as surface receptors or ligands, functioning as virulence factors, impeding host immune responses, or playing other roles in the struggle between host and microbe. In the context of the host, glycosylation drives cell–cell interactions that initiate and regulate the host response and modulates the effects of antibodies and soluble immune mediators. This perspective reports on a workshop organized jointly by the National Institute of Allergy and Infectious Diseases and the National Institute of Dental and Craniofacial Research in May 2020. The conference addressed the use of emerging glycoscience tools and resources to advance investigation of glycans and their roles in microbe-host interactions, immune-mediated diseases, and immune cell recognition and function. Future discoveries in these areas will increase fundamental scientific understanding and have the potential to improve diagnosis and treatment of infections and immune dysregulation

The crossroads of glycoscience, infection, and immunology

Advances in experimental capabilities in the glycosciences offer expanding opportunities for discovery in the broad areas of immunology and microbiology. These two disciplines overlap when microbial infection stimulates host immune responses and glycan structures are central in the processes that occur during all such encounters. Microbial glycans mediate host-pathogen interactions by acting as surface receptors or ligands, functioning as virulence factors, impeding host immune responses, or playing other roles in the struggle between host and microbe. In the context of the host, glycosylation drives cell-cell interactions that initiate and regulate the host response and modulates the effects of antibodies and soluble immune mediators. This perspective reports on a workshop organized jointly by the National Institute of Allergy and Infectious Diseases and the National Institute of Dental and Craniofacial Research in May 2020. The conference addressed the use of emerging glycoscience tools and resources to advance investigation of glycans and their roles in microbe-host interactions, immune-mediated diseases, and immune cell recognition and function. Future discoveries in these areas will increase fundamental scientific understanding and have the potential to improve diagnosis and treatment of infections and immune dysregulation

Nucleocytoplasmic transport: a thermodynamic mechanism

The nuclear pore supports molecular communication between cytoplasm and nucleus in eukaryotic cells. Selective transport of proteins is mediated by soluble receptors, whose regulation by the small GTPase Ran leads to cargo accumulation in, or depletion from the nucleus, i.e., nuclear import or nuclear export. We consider the operation of this transport system by a combined analytical and experimental approach. Provocative predictions of a simple model were tested using cell-free nuclei reconstituted in Xenopus egg extract, a system well suited to quantitative studies. We found that accumulation capacity is limited, so that introduction of one import cargo leads to egress of another. Clearly, the pore per se does not determine transport directionality. Moreover, different cargo reach a similar ratio of nuclear to cytoplasmic concentration in steady-state. The model shows that this ratio should in fact be independent of the receptor-cargo affinity, though kinetics may be strongly influenced. Numerical conservation of the system components highlights a conflict between the observations and the popular concept of transport cycles. We suggest that chemical partitioning provides a framework to understand the capacity to generate concentration gradients by equilibration of the receptor-cargo intermediary.Comment: in press at HFSP Journal, vol 3 16 text pages, 1 table, 4 figures, plus Supplementary Material include

Single-molecule imaging to characterise the transport mechanism of the Nuclear Pore Complex

In the eukaryotic cell, a large macromolecular channel, known as the Nuclear Pore Complex (NPC), mediates all molecular transport between the nucleus and cytoplasm. In recent years, single-molecule fluorescence (SMF) imaging has emerged as a powerful tool to study the molecular mechanism of transport through the NPC. More recently, techniques such as Single-Molecule Localisation Microscopy (SMLM) have enabled the spatial and temporal distribution of cargos, transport receptors and even structural components of the NPC to be determined with nanometre accuracy. In this protocol, we describe a method to study the position and/or motion of individual molecules transiting through the NPC with high spatial and temporal precision

Self-Organization of Anastral Spindles by Synergy of Dynamic Instability, Autocatalytic Microtubule Production, and a Spatial Signaling Gradient

Assembly of the mitotic spindle is a classic example of macromolecular self-organization. During spindle assembly, microtubules (MTs) accumulate around chromatin. In centrosomal spindles, centrosomes at the spindle poles are the dominating source of MT production. However, many systems assemble anastral spindles, i.e., spindles without centrosomes at the poles. How anastral spindles produce and maintain a high concentration of MTs in the absence of centrosome-catalyzed MT production is unknown. With a combined biochemistry-computer simulation approach, we show that the concerted activity of three components can efficiently concentrate microtubules (MTs) at chromatin: (1) an external stimulus in form of a RanGTP gradient centered on chromatin, (2) a feed-back loop where MTs induce production of new MTs, and (3) continuous re-organization of MT structures by dynamic instability. The mechanism proposed here can generate and maintain a dissipative MT super-structure within a RanGTP gradient