SummaryThe mechanisms by which intrinsically disordered proteins engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function, where nuclear transport receptors (NTRs) move through the NPC by binding disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Combining single-molecule fluorescence, molecular simulations, and nuclear magnetic resonance, we show that a rapidly fluctuating FG-Nup populates an ensemble of conformations that are prone to bind NTRs with near diffusion-limited on rates, as shown by stopped-flow kinetic measurements. This is achieved using multiple, minimalistic, low-affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG-Nup to maintain an unexpectedly high plasticity in its bound state. We propose that these exceptional physical characteristics enable a rapid and specific transport mechanism in the physiological context, a notion supported by single molecule in-cell assays on intact NPCs.

At the frontline of the host defense response, neutrophil antimicrobial functions have adapted to combat infections and injuries of different origins and magnitude. The release of web-like DNA structures named neutrophil extracellular traps (NETs) constitutes an important mechanism by which neutrophils prevent pathogen dissemination or deal with microorganisms of a bigger size. At the same time, nuclear and granule proteins with microbicidal activity bind to these DNA structures promoting the elimination of entrapped pathogens. However, these toxic properties may produce unwanted effects in the host, when neutrophils uncontrollably release NETs upon persistent inflammation. As a consequence, NET accumulation can produce vessel occlusion, tissue damage, and prolonged inflammation associating with the progression and exacerbation of multiple pathologic conditions. This review outlines recent advances in understanding the mechanisms of NET release and functions in sterile disease. We also discuss mechanisms of physiological regulation and the importance of neutrophil heterogeneity in NET formation and composition.

Tuberculosis (TB) is a leading cause of mortality due to infectious disease, but the factors determining disease progression are unclear. Transcriptional signatures associated with type I IFN signalling and neutrophilic inflammation were shown to correlate with disease severity in mouse models of TB. Here we show that similar transcriptional signatures correlate with increased bacterial loads and exacerbate pathology during Mycobacterium tuberculosis infection upon GM-CSF blockade. Loss of GM-CSF signalling or genetic susceptibility to TB (C3HeB/FeJ mice) result in type I IFN-induced neutrophil extracellular trap (NET) formation that promotes bacterial growth and promotes disease severity. Consistently, NETs are present in necrotic lung lesions of TB patients responding poorly to antibiotic therapy, supporting the role of NETs in a late stage of TB pathogenesis. Our findings reveal an important cytokine-based innate immune effector network with a central role in determining the outcome of M. tuberculosis infection.

We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.

The nuclear pore complex (NPC) forms a permeability barrier between the nucleus and the cytoplasm. Molecules that are able to cross this permeability barrier encounter different disordered phenylalanine glycine rich nucleoporins (FG-Nups) that act as a molecular filter and regulate the selective NPC crossing of biomolecules. In this review, we provide a current overview regarding the interaction mechanism between FG-Nups and the carrier molecules that recognize and enable the transport of cargoes through the NPC aiming to understand the general molecular mechanisms that facilitate the nucleocytoplasmic transport.