Stretching the resolution limit of atomic force microscopy

Atomic force microscopy (AFM) is unique in visualizing functional biomolecules in aqueous solution at ~1 nm resolution. By borrowing localization methods from fluorescence microscopy, AFM has been shown to discern structural domains that may be separated by only a few Ångströms

Crowding-induced phase separation of nuclear transport receptors in FG nucleoporin assemblies

The rapid (< 1 ms) transport of biological material to and from the cell nucleus is regulated by the nuclear pore complex (NPC). At the core of the NPC is a permeability barrier consisting of intrinsically disordered Phe-Gly (FG) nucleoporins (FG Nups). Various types of nuclear transport receptors (NTRs) facilitate transport by partitioning in the FG Nup assembly, overcoming the barrier by their affinity to the FG Nups, and comprise a significant fraction of proteins in the NPC barrier. In previous work Zahn et al. (2016), we revealed a universal physical behaviour in the experimentally observed binding of two well-characterized NTRs, NTF2 and the larger Importin-ᵝ, to different planar assemblies of FG Nups, with the binding behaviour defined by negative cooperativity. This was further validated by a minimal physical model that treated the FG Nups as flexible homopolymers and the NTRs as uniformly cohesive spheres. Here, we build upon our original study by first parametrizing our model to experimental data, and next predicting the effects of crowding by different types of NTRs. We show how varying the amounts of one type of NTR modulates how the other NTR penetrates the FG Nup assembly. Notably, at similar and physiologically relevant NTR concentrations, our model predicts demixed phases of NTF2 and Imp-ᵝ within the FG Nup assembly. The functional implication of NTR phase separation is that NPCs may sustain separate transport pathways that are determined by inter-NTR competition

Linear and field-independent relation between vortex core state energy and gap in Bi2Sr2CaCu2O8+d

We present a scanning tunneling spectroscopy study on quasiparticle states in vortex cores in Bi2Sr2CaCu2O8+δ. The energy of the observed vortex core states shows an approximately linear scaling with the superconducting gap in the region just outside the core. This clearly distinguishes them from conventional localized core states and is a signature of the mechanism responsible for their discrete appearance in high-temperature superconductors. The energy scaling of the vortex core states also suggests a common nature of vortex cores in Bi2Sr2CaCu2O8+δ and YBa2Cu3O7-δ. Finally, these states do not show any dependence on the applied magnetic field between 1 and 6 T

Atomic force microscopy to elucidate how peptides disrupt membranes

Atomic force microscopy is an increasingly attractive tool to study how peptides disrupt membranes. Often performed on reconstituted lipid bilayers, it provides access to time and length scales that allow dynamic investigations with nanometre resolution. Over the last decade, AFM studies have enabled visualisation of membrane disruption mechanisms by antimicrobial or host defence peptides, including peptides that target malignant cells and biofilms. Moreover, the emergence of high-speed modalities of the technique broadens the scope of investigations to antimicrobial kinetics as well as the imaging of peptide action on live cells in real time. This review describes how methodological advances in AFM facilitate new insights into membrane disruption mechanisms

Imaging live bacteria at the nanoscale: comparison of immobilisation strategies

Atomic force microscopy (AFM) provides an effective, label-free technique enabling the imaging of live bacteria under physiological conditions with nanometre precision. However, AFM is a surface scanning technique, and the accuracy of its performance requires the effective and reliable immobilisation of bacterial cells onto substrates. Here, we compare the effectiveness of various chemical approaches to facilitate the immobilisation of Escherichia coli onto glass cover slips in terms of bacterial adsorption, viability and compatibility with correlative imaging by fluorescence microscopy. We assess surface functionalisation using gelatin, poly-l-lysine, Cell-Tak™, and Vectabond®. We describe how bacterial immobilisation, viability and suitability for AFM experiments depend on bacterial strain, buffer conditions and surface functionalisation. We demonstrate the use of such immobilisation by AFM images that resolve the porin lattice on the bacterial surface; local degradation of the bacterial cell envelope by an antimicrobial peptide (Cecropin B); and the formation of membrane attack complexes on the bacterial membrane

Physical modelling of multivalent interactions in the nuclear pore complex

In the nuclear pore complex (NPC), intrinsically disordered proteins (FG Nups) along with their interactions with more globular proteins called nuclear transport receptors (NTRs) are vital to the selectivity of transport into and out of the cell nucleus. While such interactions can be modelled at different levels of coarse graining, in-vitro experimental data have been quantitatively described by minimal models that describe FG Nups as cohesive homogeneous polymers and NTRs as uniformly cohesive spheres, where the heterogeneous effects have been smeared out. By definition, these minimal models do not account for the explicit heterogeneities in FG Nup sequences, essentially a string of cohesive and non-cohesive polymer units, and at the NTR surface. Here, we develop computational and analytical models that do take into account such heterogeneity in a minimal fashion, and compare them to experimental data on single-molecule interactions between FG Nups and NTRs. Overall, we find that the heterogeneous nature of FG Nups and NTRs does play a role in determining equilibrium binding properties, but is of much greater significance when it comes to unbinding and binding kinetics. Using our models, we predict how binding equilibria and kinetics depend on the distribution of cohesive blocks in the FG Nup sequences and of the binding pockets at the NTR surface, with multivalency playing a key role. Finally, we observe that single-molecule binding kinetics has a rather minor influence on the diffusion of NTRs in polymer melts consisting of FG-Nup-like sequences

Intrinsically disordered nuclear pore proteins show ideal-polymer morphologies and dynamics

In the nuclear pore complex, intrinsically disordered nuclear pore proteins (FG Nups) form a selective barrier for transport into and out of the cell nucleus, in a way that remains poorly understood. The collective FG Nup behavior has long been conceptualized either as a polymer brush, dominated by entropic and excluded-volume (repulsive) interactions, or as a hydrogel, dominated by cohesive (attractive) interactions between FG Nups. Here we compare mesoscale computational simulations with a wide range of experimental data to demonstrate that FG Nups are at the crossover point between these two regimes. Specifically, we find that repulsive and attractive interactions are balanced, resulting in morphologies and dynamics that are close to those of ideal polymer chains. We demonstrate that this property of FG Nups yields sufficient cohesion to seal the transport barrier, and yet maintains fast dynamics at the molecular scale, permitting the rapid polymer rearrangements needed for transport events

Acoustic Immunosensing of Exosomes Using a Quartz Crystal Microbalance with Dissipation Monitoring

Exosomes are endocytic lipid-membrane bound bodies with the potential to be used as biomarkers in cancer and neurodegenerative disease. The limitations and scarcity of current exosome characterization approaches have led to a growing demand for translational techniques, capable of determining their molecular composition and physical properties in physiological fluids. Here, we investigate label-free immunosensing, using a quartz crystal microbalance with dissipation monitoring (QCM-D), to detect exosomes by exploiting their surface protein profile. Exosomes expressing the transmembrane protein CD63 were isolated by size-exclusion chromatography from cell culture media. QCM-D sensors functionalized with anti-CD63 antibodies formed a direct immunoassay toward CD63-positive exosomes in 75% v/v serum, exhibiting a limit-of-detection of 2.9 × 108 and 1.4 × 108 exosome sized particles (ESPs)/mL for frequency and dissipation response, respectively, i.e., clinically relevant concentrations. Our proof-of-concept findings support the adoption of dual-mode acoustic analysis of exosomes, leveraging both frequency and dissipation monitoring for use in bioanalytical characterization

Model inspired by nuclear pore complex suggests possible roles for nuclear transport receptors in determining its structure.

Nuclear transport receptors (NTRs) mediate nucleocytoplasmic transport via their affinity for unstructured proteins (polymers) in the nuclear pore complex (NPC). Here, we have modeled the effect of NTRs on polymeric structure in the nanopore confinement of the NPC central conduit. The model explicitly takes into account inter- and intramolecular interactions, as well as the finite size of the NTRs (∼20% of the NPC channel diameter). It reproduces various proposed scenarios for the channel structure, ranging from a central polymer condensate (selective phase) to brushlike polymer arrangements localized at the channel wall (virtual gate, reduction of dimensionality), with the transport receptors lining the polymer surface. In addition, it predicts a new structure in which NTRs become an integral part of the transport barrier by forming a cross-linked network with the unstructured proteins stretching across the pore. The model provides specific and distinctive predictions for the equilibrium spatial distributions of NTRs for these different scenarios that can be experimentally verified by, e.g., superresolution fluorescence microscopy. Moreover, it suggests mechanisms by which globular macromolecules (colloidal particles) can cause polymer-coated nanopores to switch between open and closed configurations, a possible explanation of the biological function of the NPC, and suggests potential technological applications for filtration and single-molecule sensing

Bistable collective behavior of polymers tethered in a nanopore.

Polymer-coated pores play a crucial role in nucleo-cytoplasmic transport and in a number of biomimetic and nanotechnological applications. Here we present Monte Carlo and Density Functional Theory approaches to identify different collective phases of end-grafted polymers in a nanopore and to study their relative stability as a function of intermolecular interactions. Over a range of system parameters that is relevant for nuclear pore complexes, we observe two distinct phases: one with the bulk of the polymers condensed at the wall of the pore, and the other with the polymers condensed along its central axis. The relative stability of these two phases depends on the interpolymer interactions. The existence the two phases suggests a mechanism in which marginal changes in these interactions, possibly induced by nuclear transport receptors, cause the pore to transform between open and closed configurations, which will influence transport through the pore