940 research outputs found
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
The vanishing author in computer-generated works: a critical analysis of recent Australian case law
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The use of software is ubiquitous in the creation of many copyright works, yet the requirement in copyright law that every work have a human author who engages in independent intellectual effort means that its use may prevent copyright subsistence. Several recent Australian cases have refocused attention on authorship as an essential criterion of copyright subsistence, and these cases suggest that much computer-produced output may be authorless and thus lack copyright protection. This article, the first in a two-part series, analyses how each case deals with the question of authorship of computer-produced works and why the use of software diminishes copyright protection for a significant number of computer-generated works. The article critiques the application of conventional notions of human authorship developed in the pre-computer age to modern productions and suggests alternative approaches to authorship that satisfy both the major objectives of copyright policy and the need to adapt to the computer age. The article argues that, without a broader judicial approach to authorship of computer-generated works, Parliament must remedy the lacuna in protection for these ‘authorless’ works. Possible solutions for reform are suggested. In a forthcoming article, the author comprehensively examines those reform proposals
Structures of prostacyclin synthase and its complexes with substrate-analog and inhibitor reveal a ligand-specific heme conformation change
No full textSingular equivalence and the (Fg) condition
Get PDFAbstractWe show that singular equivalences of Morita type with level between finite-dimensional Gorenstein algebras over a field preserve the (Fg) condition
De functie van kraton FG als compatibilizer in blends van nylon-6 en polypropyleen
No full textHet afstudeerproject maakte deel uit van een bij KSLA lopend onderzoek naar de werking van blok-copolymeren als compatibilizer, met name de door SHELL vervaardigde SBS en SEBS blok-copolymeren. Het voorgestelde systeem om de experimenten in het kader van de afstudeeropdracht mee uit te voeren is nylon-6/polypropyleen met kraton FG als compatibilizer. Kraton FG is een met maleinezuuranhydride gefunctionaliseerd SEBS blok-copolymeer. Kraton FG is een goede compatibilizer voor blends van nylon-6 en polypropyleen. De grensvlakspanning werd verlaagd, waardoor een fijnere dispersie verkregen werd. De slagvastheid nam toe. Het is niet goed mogelijk om een verband te leggen tussen de verlaging van de grensvlakspanning en de verlaging van de deeltjesgrootte. In dit onderzoek werd de grensvlakspanning in een 'spinning drop' met behulp van enkele methoden bepaald. Bij SHELL is een methode ontwikkeld, die aanzienlijk lagere grensvlakspanningen leverde dan de andere methoden. De tijdsduur van een meting volgens deze methode is ook veel lager (1- 2 uur in plaats van 4-6 uur). De toename van de slagvastheid was het grootst in blends met 25% disperse fase (6.25% kraton FG). Dit was het maximum in de serie blends, die tijdens dit onderzoek getest werden. Om de verbetering van de slagvastheid ten gevolge van de aanwezigheid van kraton FG te begrijpen, moet ook naar de eigenschappen van de matrix fase gekeken worden. De hechting tussen beide fasen zal door de aanwezigheid van kraton FG niet verbeterd worden. Dit is ook niet nodig, daar 'shear yielding' het voornaamste 'toughening' mechanisme in nylon-6 is. Door de reactie tussen kraton FG en nylon-6 zal de viscositeit van de matrix-fase toenemen. Deze verhoging van de viscositeit zal van invloed zijn op het dispersieproces. Dit verschijnsel is nog niet goed bekend, daar het onderzoek naar compatibilizers vooral gericht is op het grensvlak tussen de matrix en disperse fase (grensvlakspanning, adhesie). Door de aanwezigheid van compatibilizers wordt coalescentie van de deeltjes tegengegaan. De verklaring, die hiervoor gegeven wordt, is kinetisch van aard. Het is goed mogelijk dat coalescentie van twee deeltjes thermodynamisch ongunstig is ten gevolge van de aanwezigheid van compatibilizers aan het grensvlak.Applied Science
A designer FG-Nup that reconstitutes the selective transport barrier of the nuclear pore complex
Get PDFNuclear Pore Complexes (NPCs) regulate bidirectional transport between the nucleus and the cytoplasm. Intrinsically disordered FG-Nups line the NPC lumen and form a selective barrier, where transport of most proteins is inhibited whereas specific transporter proteins freely pass. The mechanism underlying selective transport through the NPC is still debated. Here, we reconstitute the selective behaviour of the NPC bottom-up by introducing a rationally designed artificial FG-Nup that mimics natural Nups. Using QCM-D, we measure selective binding of the artificial FG-Nup brushes to the transport receptor Kap95 over cytosolic proteins such as BSA. Solid-state nanopores with the artificial FG-Nups lining their inner walls support fast translocation of Kap95 while blocking BSA, thus demonstrating selectivity. Coarse-grained molecular dynamics simulations highlight the formation of a selective meshwork with densities comparable to native NPCs. Our findings show that simple design rules can recapitulate the selective behaviour of native FG-Nups and demonstrate that no specific spacer sequence nor a spatial segregation of different FG-motif types are needed to create selective NPCs.</p
Analytical and FE Modeling of FG Beams Based on A Refined Shear Deformable Beam Theory for Static and Dynamic Analyses of FG BeamsWith Thermoelastic Coupling
Get PDFThe static and dynamic thermoelastic analyses of the beams made of functionally graded materials (FGMs) are presented in this paper. Based on the refined third-order shear deformation beam theory proposed by the senior author and the variational principle, the governing equations of FG beams are deduced. The influence of temperature on Young’s modulus and coefficients of thermal expansion is taken into account when FG beams are subjected to thermal loading. The resulting governing equations are a system of the eighth-order differential equations in terms of displacement variables, and the thermoelastic coupling is included in the equations. An accurate and reliable two-noded beam element is developed for the bending and free vibration analysis of FG beams by employing the refined thirdorder shear deformation beam theory and the quasi-conforming element technique. Several typical examples of FG beams are solved using the present FG beam element to show the effects of the material distribution and thermal loading on the defections, stresses and natural frequencies of FG beams. The accuracy of both the analytical solutions and numerical results given by the proposed models are validated against the results reported in the literature or the 2D finite element results solved by the authors. The results show that the present models are capable of yielding not only accurate displacements but also accurate stresses and higher-order frequencies of free vibration for the FG beams with thermoelastic coupling
Quantitative flow cytometric analysis of FG and thy-1 labeled RGCs.
No full text<p>(A) Effect of NMDA on survival rates of different size RGCs and total RGCs compared to the control group (n = 6). Total RGCs were counted using a hemacytometer and the percentages of different size RGCs in total retinal cells were determined by flow cytometry. The numbers of different size RGCs were calculated according to the above results. (B) Quantitative analysis of FG intensities of different size RGCs following intravitreal NMDA injection (n = 6). (C) Quantitative analysis of thy-1 intensities of different size RGCs following intravitreal NMDA injection (n = 6). FG and thy-1 intensities were expressed as geometric means calculated by CellQuest Pro software. Data were presented as mean ± standard error of the mean. Differences between the control and NMDA groups were analyzed using the Student's <i>t</i>-test. *P<0.05, **P<0.01 and ***P<0.001.</p
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