250 research outputs found
Nanomechanics of confined polymer systems
Get PDFPolymers anchored to surfaces play an important role in nature and technology, and regulate diverse interfacial phenomena in areas such as tribology and colloidal stability. Polymers grafted to surfaces at high density form elongated “brushes” with characteristic lengths much larger than free coils in solution. These brushes can reduce interfacial friction and wear as well as impart fouling resistance to surfaces. In light of these functionalities it is important to understand the behaviour of surface-grafted polymers at the molecular and nanoscopic level. An emerging area of interest are polymers attached to nanopores. Theoretical studies predict interesting morphologies and dynamics of such confined brushes in and around nanopores, but nanopore environments have been difficult to study experimentally. In this thesis a unique polymer-functionalized nanopore-like experimental system is presented, functionalized with poly(ethylene glycol) (PEG). Atomic force microscopy (AFM) is employed to probe the PEG brushes with nanometre spatial precision and sub-nanonewton force sensitivity, revealing novel dynamics depending on the local grafting position of PEG with respect to the nanopore geometry. Further, AFM is used together with fluorescence microscopy to show how polymer–protein interactions can be used together with the anti-fouling property of PEG to sort specific biomolecules from complex biological fluids to nanoscale targets. This shows a way how to confer biological recognition and specificity to synthetic nanoscale systems which is important for biosensing and bioseparation applications
Polyphony and the anxiety of influence in the fiction of Henry James
Get PDFJames's fiction, especially in the Middle Phase, centres
on the figure of the artist and is characterized by, the two
interrelated aspects which previous criticism has largely
overlooked: the Bakhtinian 'polyphonic' -creation of
'author-thinkers'; and the conflict between ephebes and
precursors, for which Harold-Bloom's concept of 'the-anxiety of
influence' is the most illuminating model. Polyphony is the
narrative mode, and influence is the intra-artistic, theme.
These, as the Introduction to the thesis makes clear, are
rehearsed in James's inaugural novel, Roderick Hudson. Rowland
Mallet is an author-thinker, and his failure is caused by
authorial limitations. His monologism -is impaired by his
mistaking empathy for the authorial sympathy. Likewise,
Hudson's failure does not arise from a mercurial temperament,
but from a polyphonic shortcoming: not possessing the power of
fiction to contain the fiction of power in, his mentor. And the
relationships among the three artists - Gloriani, Hudson and
Singleton - perfectly exemplify the Bloomian-theme. It is these
two concepts, polyphony and influence, which are the major
preoccupation in the Middle Phase; as, the works chosen
demonstrate. These are a novella, a novel, and a number of
short stories all of which have been unjustifiably neglected.
Chapter One, on The Aspern Papers, argues that Tina Bordereau,
far from being, the artless victim seen by many critics,
actually challenges and defeats the narrator by the very form
of her narrative. Her 'realist' discourse undermines his
language of 'romance', and shows up its internal unstability.
Chapter Two is an extensive study of the critical reception of
The Tragic Muse. The most common areas of critical attention
have been its contemporary topicality, its relation to previous
novels on similar themes, and the possible genealogy of Gabriel
Nash. Those have all missed the core of the work. - Chapter Three
demonstrates how polyphony and the anxiety of influence make
the novel what it really is. Influence arises from the
juxtaposition of, and the wrestling between, artistic ephebes
and their precursors (Nick and Nash,, Miriam and Madame Carre).
The dialogic quality defined by Bakhtin is crucial to the
proper, and even-handed, characterization of all, the conflicts
in the novel. And since most of James's tales in the eighties
and nineties -are about 'masters - and acolytes, the anxiety of
influence remains central. Chapter Four is a study of 'The
Author of Beltraffiol' and 'The Lesson of the Master'. Again the
characters' manipulations are a crucial focus in a way that
G6rard Genette's terminology helps to illuminate. The fact that
the ephebe is the author-thinker emphasizes the inextricability
of the Bakhtinian and the Bloomian in James. Just as
polyphony offers a different focus for explicating the poetics
of James's fiction; so the ephebal conflict provides the basis
for a fresh perception of James's own artistic struggle
Reduction of dimensionality in Karyopherinβ1 mediated transport on FG domains
Get PDFMany molecular transport processes in living cells proceed by facilitated diffusion in two dimensions instead of three, but how this process works remains poorly understood. Known as “reduction of dimensionality” (ROD), this phenomenon has been implicated to underlie the transport of proteins through nuclear pore complexes (NPCs).
NPCs are biological nanomachines that regulate the selective exchange of macromolecular cargoes between the cytoplasm and nucleus in living cells. Small molecules diffuse freely through the NPC, whereas macromolecules >~5 nm in size are withheld. Access is limited to cargo-carrying transport receptors (karyopherins or Kaps, e.g. Kapß1), which interact with several intrinsically disordered Phe-Gly (FG)-repeat rich domains (i.e. FG domains) that pave the central pore. As each Kapß1 molecule contains ~10 hydrophobic pockets that bind FG repeats, Kap-FG domain binding involves highly multivalent interactions, which are generally known to impart a strong avidity that enhances stability and specificity. Consequently, in vitro studies have revealed very stable Kap-FG domain complexes. However, this is paradoxical in the context of the NPC, because the high Kapß1-FG domain binding affinities in the submicromolar range predict slow dissociation rates that contradict the short Kap-NPC dwell times measured in vivo (~5 ms). As this implies, Kap-FG domain binding ought to be sufficiently strong to ensure selectivity, but also weak enough to promote fast translocation through the NPC. However, an explanation as to how Kap-FG domain interaction balances the tradeoff between mobility and specificity during nucleocytoplasmic transport (NCT) is still lacking.
In the work presented here, this discrepancy is addressed in vitro using optical trapping-based photonic force microscopy (PFM). By measuring the thermal fluctuations of Kap-functionalized colloidal probes in contact with a surface grafted FG domain layer, it was found that Kap-FG interactions per se attenuate diffusive motion due to strong specific binding. This can be controlled by varying the amount of free Kaps in solution, which leads to differential behavior ranging from highly constrained to near-passive diffusion that is attributed to diminishing multivalent interactions between the Kap-probe and the FG domain layer. Measurements using surface plasmon resonance are consistent with this interpretation and show that a reduction of free FG-binding sites follows from a concentration-dependent increase in the occupancy of soluble Kapß1 molecules within the FG domain layer.
With the optical trap switched off, the probes exhibited two-dimensional diffusion at physiological Kap concentrations. The dissertation explains how multivalent interactions balance binding affinity and Kap-facilitated mobility on FG domains, leading to “reduction of dimensionality” in selective transport processes. This has implications for NCT, where a ROD-based scenario was proposed in which Kaps can diffuse in two dimensions along a layer of FG domains lining the central pore. Although this has not been validated in vivo, the physical display of Kap-facilitated two-dimensional diffusion on FG domains indicates that ROD can play a functional role in expediting selective transport through biological NPCs.
The importance and relevance of the work lie both in the understanding of multivalent interactions and multivalency-regulated transport processes in biological systems, as well as in breaking ground for the development of controlled reduced dimensional diffusion and controlled motion in artificial systems. On a more technical note, this work demonstrates the use of PFM in accessing particle diffusivity in the presence of biochemical interactions at biointerfaces
Kap-Centric control of nuclear pores based on promiscuous binding to FG nucleoporins
Get PDFNuclear pore complexes (NPCs) are remarkable molecular machines that perforate the nuclear envelope (NE) in eukaryotic cells and mediate the rapid bidirectional traffic of hundreds of proteins, ribonucleoproteins, and metabolites across the nuclear envelope. Their enormous structure is composed of multiple copies of 30 different proteins (Nups) that add up to 60 – 120 MDa of mass depending on the organism. Each NPC contains a 50 nm-diameter central channel through which only molecules smaller than ~40 kDa or ~5 nm in size can diffuse passively. The movement of larger molecules is impaired by a permeability barrier generated by ~200 partly intrinsically disordered phenylalanine-glycine (FG)-rich nucleoporins (FG Nups) that are tethered to the NPC transport channel surface. These FG Nups interact promiscuously with nuclear transport receptors (NTRs), such as karyopherins (Kaps; e.g. Kap-beta1) or NTF2, that mediate rapid trafficking of cargoes.
Given that the number of FG repeats per FG Nup also varies from 5 to ~50, NTR-FG Nup binding involves highly multivalent interactions, which are generally known to impart a strong avidity that enhances stability and specificity. However, this is paradoxical in the context of the NPC, because the high submicromolar Kap-beta1-FG domain binding affinities predict slow off rates (given a diffusion-limited on rate) that contradict the rapid (~5 ms) in vivo dwell time. As this implies, Kap-FG binding ought to be sufficiently strong to ensure selectivity but also weak enough to promote fast translocation through the NPC. Nonetheless, an explanation as to how promiscuous binding of FG Nups to NTRs is balanced against the mechanistic control of the FG domain barrier is still lacking.
The purpose of my work was to investigate FG Nup-NTR binding promiscuity and multivalency by measuring the interaction kinetics, binding affinity and in situ associated conformational changes in Nsp1p FG domains when binding NTF2 and Kap-beta1, both separately and together. Experimentally, this was achieved by using a novel surface plasmon resonance technique to correlate in situ mechanistic changes (molecular occupancy and conformational changes) with FG Nup-NTR binding.
The obtained results show that surface-tethered Nsp1p FG domains form molecular brushes at physiological conditions. Kap-beta1 binding provokes brush extension while partitioning into a fast and slow kinetic phase, where the latter may form an integral part of the FG domain barrier. In contrast, NTF2 binding to pristine Nsp1p layers induced collapse, but not under competing interactions from Kap-beta1. Therefore, promiscuous binding of NTF2 to Kap-beta1-preloaded Nsp1p attenuates NTF2 towards higher off rates and more transient interactions.
My work demonstrates that promiscuous binding of NTRs to FG Nups ought to influence nucleocytoplasmic transport. This depends on the concentration, size and binding strength of each NTR. Indeed, some form of hierarchy may exist between different NTRs such that their relative concentrations may impact NPC barrier function. This interpretation departs from the conventional view that the FG Nups alone form the NPC permeability barrier. Rather I conclude that concentrating NTRs in the NPC transport channel also contributes to generating crowding-based selective barrier function of the pore
Phosphorylation but Not Oligomerization Drives the Accumulation of Tau with Nucleoporin Nup98
Get PDFTau is a neuronal protein that stabilizes axonal microtubules (MTs) in the central nervous system. In Alzheimer’s disease (AD) and other tauopathies, phosphorylated Tau accumulates in intracellular aggregates, a pathological hallmark of these diseases. However, the chronological order of pathological changes in Tau prior to its cytosolic aggregation remains unresolved. These include its phosphorylation and detachment from MTs, mislocalization into the somatodendritic compartment, and oligomerization in the cytosol. Recently, we showed that Tau can interact with phenylalanine-glycine (FG)-rich nucleoporins (Nups), including Nup98, that form a diffusion barrier inside nuclear pore complexes (NPCs), leading to defects in nucleocytoplasmic transport. Here, we used surface plasmon resonance (SPR) and bio-layer interferometry (BLI) to investigate the molecular details of Tau:Nup98 interactions and determined how Tau phosphorylation and oligomerization impact the interactions. Importantly, phosphorylation, but not acetylation, strongly facilitates the accumulation of Tau with Nup98. Oligomerization, however, seems to inhibit Tau:Nup98 interactions, suggesting that Tau-FG Nup interactions occur prior to oligomerization. Overall, these results provide fundamental insights into the molecular mechanisms of Tau-FG Nup interactions within NPCs, which might explain how stress-and disease-associated posttranslational modifications (PTMs) may lead to Tau-induced nucleocytoplasmic transport (NCT) failure. Intervention strategies that could rescue Tau-induced NCT failure in AD and tauopathies will be further discussed
Nucleocytoplasmic Transport: A Paradigm for Molecular Logistics in Artificial Systems
Get PDFArtificial organelles, molecular factories and nanoreactors are membrane-bound systems envisaged to exhibit cell-like functionality. These constitute liposomes, polymersomes or hybrid lipo-polymersomes that display different membrane-spanning channels and/or enclose molecular modules. To achieve more complex functionality, an artificial organelle should ideally sustain a continuous influx of essential macromolecular modules (i.e. cargoes) and metabolites against an outflow of reaction products. This would benefit from the incorporation of selective nanopores as well as specific trafficking factors that facilitate cargo selectivity, translocation efficiency, and directionality. Towards this goal, we describe how proteinaceous cargoes are transported between the nucleus and cytoplasm by nuclear pore complexes and the biological trafficking machinery in living cells (i.e. nucleocytoplasmic transport). On this basis, we discuss how biomimetic control may be implemented to selectively import, compartmentalize and accumulate diverse macromolecular modules against concentration gradients in artificial organelles
Gate-crashing the nuclear pore complex
No full textAs a third in a series of MD simulations investigating the binding dynamics between nuclear transport receptors and FG-repeats, Isgro and Schulten (2007b) unveil that close, physical intimacy between partners is likely to ensure a hassle-free passage through the nuclear pore complex
Surface-modified elastomeric nanofluidic devices for single nanoparticle trapping
No full textAbstract Our work focuses on the development of simpler and effective production of nanofluidic devices for high-throughput charged single nanoparticle trapping in an aqueous environment. Single nanoparticle confinement using electrostatic trapping has been an effective approach to study the fundamental properties of charged molecules under a controlled aqueous environment. Conventionally, geometry-induced electrostatic trapping devices are fabricated using SiOx-based substrates and comprise nanochannels imbedded with nanoindentations such as nanopockets, nanoslits and nanogrids. These geometry-induced electrostatic trapping devices can only trap negatively charged particles, and therefore, to trap positively charged particles, modification of the device surface is required. However, the surface modification process of a nanofluidic device is cumbersome and time consuming. Therefore, here, we present a novel approach for the development of surface-modified geometry-induced electrostatic trapping devices that reduces the surface modification time from nearly 5 days to just a few hours. We utilized polydimethylsiloxane for the development of a surface-modified geometry-induced electrostatic trapping device. To demonstrate the device efficiency and success of the surface modification procedure, a comparison study between a PDMS-based geometry-induced electrostatic trapping device and the surface-modified polydimethylsiloxane-based device was performed. The device surface was modified with two layers of polyelectrolytes (1: poly(ethyleneimine) and 2: poly(styrenesulfonate)), which led to an overall negatively charged surface. Our experiments revealed the presence of a homogeneous surface charge density inside the fluidic devices and equivalent trapping strengths for the surface-modified and native polydimethylsiloxane-based geometry-induced electrostatic trapping devices. This work paves the way towards broader use of geometry-induced electrostatic trapping devices in the fields of biosensing, disease diagnosis, molecular analysis, fluid quality control and pathogen detection
Non-interacting molecules as innate structural probes in surface plasmon resonance
No full textDetermining the structural parameters of a molecular layer remains an unresolved problem in surface plasmon resonance (SPR). Given that molecular form and function are intimately coupled, a breakthrough in this area could be of considerable benefit to the study of protein and/or polymer-decorated material interfaces that are ubiquitous in biology and technology. Here, we describe how noninteracting molecules function as innate structural probes that "feel" the intrinsic exclusion volume of a surface-tethered molecular layer in SPR. Importantly, this is noninvasive and provides a means to bypass the refractive index (RI) constraint that convolutes and hinders SPR thickness measurements. To show proof-of-concept, we use BSA molecules in solution to measure the thicknesses of polyethylene glycol (PEG) molecular brushes as a function of molecular weight. The SPR-acquired brush thicknesses scale with PEG hydrodynamic diameter and are in good agreement with atomic force microscopy force-distance measurements. Theoretical treatments that account for changes in the evanescent field decay length at the metal-dielectric interface indicate that the method is most appropriate for low RI layers with an estimated maximal error of ±15% in the thickness due to the RI constraint. Such in situ thickness measurements can be easily incorporated into routine SPR binding assays for investigating mesoscopic structure-function correlations of diverse molecular layers (i.e., biointerfaces)
Towards reconciling structure and function of the nuclear pore complex
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