Integrative spatiotemporal map of nucleocytoplasmic transport.

The Nuclear Pore Complex (NPC) facilitates rapid and selective nucleocytoplasmic transport of molecules as large as ribosomal subunits and viral capsids. It is not clear how key emergent properties of this transport arise from the system components and their interactions. To address this question, we constructed an integrative coarse-grained Brownian dynamics model of transport through a single NPC, followed by coupling it with a kinetic model of Ran-dependent transport in an entire cell. The microscopic model parameters were fitted to reflect experimental data and theoretical information regarding the transport, without making any assumptions about its emergent properties. The resulting reductionist model is validated by reproducing several features of transport not used for its construction, such as the morphology of the central transporter, rates of passive and facilitated diffusion as a function of size and valency, in situ radial distributions of pre-ribosomal subunits, and active transport rates for viral capsids. The model suggests that the NPC functions essentially as a virtual gate whose flexible phenylalanine-glycine (FG) repeat proteins raise an entropy barrier to diffusion through the pore. Importantly, this core functionality is greatly enhanced by several key design features, including 'fuzzy' and transient interactions, multivalency, redundancy in the copy number of FG nucleoporins, exponential coupling of transport kinetics and thermodynamics in accordance with the transition state theory, and coupling to the energy-reliant RanGTP concentration gradient. These design features result in the robust and resilient rate and selectivity of transport for a wide array of cargo ranging from a few kilodaltons to megadaltons in size. By dissecting these features, our model provides a quantitative starting point for rationally modulating the transport system and its artificial mimics.

Predicting the structural basis of targeted protein degradation by integrating molecular dynamics simulations with structural mass spectrometry.

Targeted protein degradation (TPD) is a promising approach in drug discovery for degrading proteins implicated in diseases. A key step in this process is the formation of a ternary complex where a heterobifunctional molecule induces proximity of an E3 ligase to a protein of interest (POI), thus facilitating ubiquitin transfer to the POI. In this work, we characterize 3 steps in the TPD process. (1) We simulate the ternary complex formation of SMARCA2 bromodomain and VHL E3 ligase by combining hydrogen-deuterium exchange mass spectrometry with weighted ensemble molecular dynamics (MD). (2) We characterize the conformational heterogeneity of the ternary complex using Hamiltonian replica exchange simulations and small-angle X-ray scattering. (3) We assess the ubiquitination of the POI in the context of the full Cullin-RING Ligase, confirming experimental ubiquitinomics results. Differences in degradation efficiency can be explained by the proximity of lysine residues on the POI relative to ubiquitin.

Biological motion elicits between-person Inhibition of Return in temporal and spatial movement parameters.

The present study was designed to gain a deeper understanding of the processing of biological motion stimuli. To this end, we investigated if the inhibition of return (IoR) effect emerges in initiation times and trajectories of pointing movements to targets in left and right space where the preceding cues were pointing movement of a human model or a dot with the same biological motion. Targets were randomly presented in the same or opposite side from the direction of the motion cue. It was hypothesised that the visuomotor system should resonate with the biological motion of a dot, but that the human model should exaggerate the effect. Thus, the human model should trigger stronger attention shifts compared with the dot model and lead to more robust IoR effects in both spatial (movement) and temporal parameters of the observer's pointing responses. Initiation times and the spatial parameters (angle of the hand trajectory) of the pointing movements were analysed. Results indicate that facilitation and IoR effects triggered by human and dot stimuli did not differ. Based on these findings, it seems that the crucial feature of motion cues that generate shifts in attention is biological motion, rather than human appearance per se.

Importance of suberin biopolymer in plant function, contributions to soil organic carbon and in the production of bio-derived energy and materials.

Suberin is a hydrophobic biopolymer of significance in the production of biomass-derived materials and in biogeochemical cycling in terrestrial ecosystems. Here, we describe suberin structure and biosynthesis, and its importance in biological (i.e., plant bark and roots), ecological (soil organic carbon) and economic (biomass conversion to bioproducts) contexts. Furthermore, we highlight the genomics and analytical approaches currently available and explore opportunities for future technologies to study suberin in quantitative and/or high-throughput platforms in bioenergy crops. A greater understanding of suberin structure and production in lignocellulosic biomass can be leveraged to improve representation in life cycle analysis and techno-economic analysis models and enable performance improvements in plant biosystems as well as informed crop system management to achieve economic and environmental co-benefits.

Self-bias effect: movement initiation to self-owned property is speeded for both approach and avoidance actions.

Recall of, and physical interaction with, self-owned items is privileged over items owned by other people (Constable et al. in Cognition 119(3):430-437, 2011; Cunningham et al. in Conscious Cognit 17(1):312-318, 2008). Here, we investigate approach (towards the item), compared with avoidance (away from the item) movements to images of self- and experimenter-owned items. We asked if initiation time and movement duration of button-press approach responses to self-owned items are associated with a systematic self-bias (overall faster responses), compared with avoidance movements, similar to findings of paradigms investigating affective evaluation of (unowned) items. Participants were gifted mugs to use, and after a few days they completed an approach-avoidance task (Chen and Bargh in Pers Soc Psychol Bull 25(2):215-224, 1999; Seibt et al. in J Exp Soc Psychol 44:713-720, 2008; Truong et al. in J Exp Psychol Hum Percept Perform 42(3), 375-385, 2016) to images of their own or the experimenter's mug, using either congruent or incongruent movement direction mappings. There was a self-bias effect for initiation time to the self-owned mug, for both congruent and incongruent mappings, and for movement duration in the congruent mapping. The effect was abolished in Experiment 2 when participants responded based on a shape on the handle rather than mug ownership. We speculate that ownership status requires conscious processing to modulate responses. Moreover, ownership status judgements and affective evaluation may employ different mechanisms.

Analysis of Multivalent IDP Interactions: Stoichiometry, Affinity, and Local Concentration Effect Measurements.

Nuclear magnetic resonance (NMR) titration and isothermal titration calorimetry can be combined to provide an assessment of how multivalent intrinsically disordered protein (IDP) interactions can involve enthalpy-entropy balance. Here, we describe the underlying technical details and additional methods, such as dynamic light scattering analysis, needed to assess these reactions. We apply this to a central interaction involving the disordered regions of phe-gly nucleoporins (FG-Nups) that contain multiple phenylalanine-glycine repeats which are of particular interest, as their interactions with nuclear transport factors (NTRs) underlie the paradoxically rapid yet also highly selective transport of macromolecules mediated by the nuclear pore complex (NPC). These analyses revealed that a combination of low per-FG motif affinity and the enthalpy-entropy balance prevents high-avidity interaction between FG-Nups and NTRs while the large number of FG motifs promotes frequent FG-NTR contacts, resulting in enhanced selectivity.

To have and to hold: embodied ownership is established in early childhood.

We investigated whether embodied ownership is evident in early childhood. To do so, we gifted a drinking bottle to children (aged 24-48 months) to use for 2 weeks. They returned to perform reach-grasp-lift-replace actions with their own or the experimenter's bottle while we recorded their movements using motion capture. There were differences in motor interactions with self- vs experimenter-owned bottles, such that children positioned self-owned bottles significantly closer to themselves compared with the experimenter's bottle. Age did not modulate the positioning of the self-owned bottle relative to the experimenter-owned bottle. In contrast, the pattern was not evident in children who selected one of the two bottles to keep only after the task was completed, and thus did not 'own' it during the task (Experiment 2). These results extend similar findings in adults, confirming the importance of ownership in determining self-other differences and provide novel evidence that object ownership influences sensorimotor processes from as early as 2 years of age.

Biological motion and animacy belief induce similar effects on involuntary shifts of attention.

Biological motion is salient to the human visual and motor systems and may be intrinsic to the perception of animacy. Evidence for the salience of visual stimuli moving with trajectories consistent with biological motion comes from studies showing that such stimuli can trigger shifts of attention in the direction of that motion. The present study was conducted to determine whether or not top-down beliefs about animacy can modify the salience of a nonbiologically moving stimulus to the visuomotor system. A nonpredictive cuing task was used in which a white dot moved from a central location toward a left- or right-sided target placeholder. The target randomly appeared at either location 200, 600, or 1,300 ms after the motion onset. Five groups of participants experienced different stimulus conditions: (1) biological motion, (2) inverted biological motion, (3) nonbiological motion, (4) animacy belief (paired with nonbiological motion), and (5) computer-generated belief (paired with nonbiological motion). Analysis of response times revealed that the motion in the biological motion and animacy belief groups, but not in the inverted and nonbiological motion groups, affected processing of the target information. These findings indicate that biological motion is salient to the visual system and that top-down beliefs regarding the animacy of the stimulus can tune the visual and motor systems to increase the salience of nonbiological motion.

Top-down attentional factors modulate action priming in reach-to-grasp action.

Previous studies report that viewing exaggerated, high-lifting reaches (versus direct reaches) primes higher vertical deviation in wrist trajectory in the observer's subsequent reaches (trajectory priming), but it is unclear to what extent this effect depends upon task instructions relevant to top-down attention. In two experiments, participants were instructed to gaze at a dot presented on a large monitor for a colour-change go signal that cued them to execute a direct reach to a target. In the background, the monitor also displayed life-sized films of a human model. The films were of the model either remaining still or reaching to grasp a target with either a direct trajectory or an exaggerated, high-lifting trajectory. When the dot traced the human model's wrist throughout her movement, a robust trajectory priming effect emerged. When the dot remained stationary in a central location but the human model reached in the background, the human model's trajectory did not alter the participants' trajectories. Finally, when the dot traced exaggerated and direct trajectories and the human model remained stationary, the dot's movement produced an attenuated, non-significant trajectory priming effect. These findings show that top-down attentional factors modulate trajectory priming. In addition, a moving non-human stimulus does not produce the same degree of action priming when contextual factors make salient its independence of human agency and/or intention.

Deciphering the "Fuzzy" Interaction of FG Nucleoporins and Transport Factors Using Small-Angle Neutron Scattering.

The largely intrinsically disordered phenylalanine-glycine-rich nucleoporins (FG Nups) underline a selectivity mechanism that enables the rapid translocation of transport factors (TFs) through the nuclear pore complexes (NPCs). Conflicting models of NPC transport have assumed that FG Nups undergo different conformational transitions upon interacting with TFs. To selectively characterize conformational changes in FG Nups induced by TFs we performed small-angle neutron scattering (SANS) with contrast matching. Conformational-ensembles derived from SANS data indicated an increase in the overall size of FG Nups is associated with TF interaction. Moreover, the organization of the FG motif in the interacting state is consistent with prior experimental analyses defining that FG motifs undergo conformational restriction upon interacting with TFs. These results provide structural insights into a highly dynamic interaction and illustrate how functional disorder imparts rapid and selective FG Nup-TF interactions.

Thermodynamic characterization of the multivalent interactions underlying rapid and selective translocation through the nuclear pore complex.

Intrinsically disordered proteins (IDPs) play important roles in many biological systems. Given the vast conformational space that IDPs can explore, the thermodynamics of the interactions with their partners is closely linked to their biological functions. Intrinsically disordered regions of Phe-Gly nucleoporins (FG Nups) that contain multiple phenylalanine-glycine repeats are of particular interest, as their interactions with transport factors (TFs) underlie the paradoxically rapid yet also highly selective transport of macromolecules mediated by the nuclear pore complex. Here, we used NMR and isothermal titration calorimetry to thermodynamically characterize these multivalent interactions. These analyses revealed that a combination of low per-FG motif affinity and the enthalpy-entropy balance prevents high-avidity interaction between FG Nups and TFs, whereas the large number of FG motifs promotes frequent FG-TF contacts, resulting in enhanced selectivity. Our thermodynamic model underlines the importance of functional disorder of FG Nups. It helps explain the rapid and selective translocation of TFs through the nuclear pore complex and further expands our understanding of the mechanisms of "fuzzy" interactions involving IDPs.

Effects of FGFR2 kinase activation loop dynamics on catalytic activity.

The structural mechanisms by which receptor tyrosine kinases (RTKs) regulate catalytic activity are diverse and often based on subtle changes in conformational dynamics. The regulatory mechanism of one such RTK, fibroblast growth factor receptor 2 (FGFR2) kinase, is still unknown, as the numerous crystal structures of the unphosphorylated and phosphorylated forms of the kinase domains show no apparent structural change that could explain how phosphorylation could enable catalytic activity. In this study, we use several enhanced sampling molecular dynamics (MD) methods to elucidate the structural changes to the kinase's activation loop that occur upon phosphorylation. We show that phosphorylation favors inward motion of Arg664, while simultaneously favoring outward motion of Leu665 and Pro666. The latter structural change enables the substrate to bind leading to its resultant phosphorylation. Inward motion of Arg664 allows it to interact with the γ-phosphate of ATP as well as the substrate tyrosine. We show that this stabilizes the tyrosine and primes it for the catalytic phosphotransfer, and it may lower the activation barrier of the phosphotransfer reaction. Our work demonstrates the value of including dynamic information gleaned from computer simulation in deciphering RTK regulatory function.

Culture modulates implicit ownership-induced self-bias in memory.

The relation of incoming stimuli to the self implicitly determines the allocation of cognitive resources. Cultural variations in the self-concept shape cognition, but the extent is unclear because the majority of studies sample only Western participants. We report cultural differences (Asian versus Western) in ownership-induced self-bias in recognition memory for objects. In two experiments, participants allocated a series of images depicting household objects to self-owned or other-owned virtual baskets based on colour cues before completing a surprise recognition memory test for the objects. The 'other' was either a stranger or a close other. In both experiments, Western participants showed greater recognition memory accuracy for self-owned compared with other-owned objects, consistent with an independent self-construal. In Experiment 1, which required minimal attention to the owned objects, Asian participants showed no such ownership-related bias in recognition accuracy. In Experiment 2, which required attention to owned objects to move them along the screen, Asian participants again showed no overall memory advantage for self-owned items and actually exhibited higher recognition accuracy for mother-owned than self-owned objects, reversing the pattern observed for Westerners. This is consistent with an interdependent self-construal which is sensitive to the particular relationship between the self and other. Overall, our results suggest that the self acts as an organising principle for allocating cognitive resources, but that the way it is constructed depends upon cultural experience. Additionally, the manifestation of these cultural differences in self-representation depends on the allocation of attentional resources to self- and other-associated stimuli.

Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex.

Nucleocytoplasmic transport is mediated by the interaction of transport factors (TFs) with disordered phenylalanine-glycine (FG) repeats that fill the central channel of the nuclear pore complex (NPC). However, the mechanism by which TFs rapidly diffuse through multiple FG repeats without compromising NPC selectivity is not yet fully understood. In this study, we build on our recent NMR investigations showing that FG repeats are highly dynamic, flexible, and rapidly exchanging among TF interaction sites. We use unbiased long timescale all-atom simulations on the Anton supercomputer, combined with extensive enhanced sampling simulations and NMR experiments, to characterize the thermodynamic and kinetic properties of FG repeats and their interaction with a model transport factor. Both the simulations and experimental data indicate that FG repeats are highly dynamic random coils, lack intrachain interactions, and exhibit significant entropically driven resistance to spatial confinement. We show that the FG motifs reversibly slide in and out of multiple TF interaction sites, transitioning rapidly between a strongly interacting state and a weakly interacting state, rather than undergoing a much slower transition between strongly interacting and completely noninteracting (unbound) states. In the weakly interacting state, FG motifs can be more easily displaced by other competing FG motifs, providing a simple mechanism for rapid exchange of TF/FG motif contacts during transport. This slide-and-exchange mechanism highlights the direct role of the disorder within FG repeats in nucleocytoplasmic transport, and resolves the apparent conflict between the selectivity and speed of transport.

Top-down control and directed attention in self-reference effects: Goal-directed movements and the SAN.

We focus on Humphreys and Sui's postulations that self-reference effects are not necessarily pre-attentive, and the self and top-down attention interact in the SAN. If so, top-down factors (goal-relevance, directed attention) should interact with self-reference effects. Our pilot data from unspeeded reach-to-grasp actions show differences in trajectories when reaching toward self- or other-relevant objects. We speculate that goal-directed actions are suited to studying the top-down control in self-reference effects. Because goal-directed action paradigms allow broad scope for modulating attention and top-down control, they will be useful for disambiguating the roles of directed attention, inhibition, and (social) context.

The molecular mechanism of nuclear transport revealed by atomic-scale measurements.

Nuclear pore complexes (NPCs) form a selective filter that allows the rapid passage of transport factors (TFs) and their cargoes across the nuclear envelope, while blocking the passage of other macromolecules. Intrinsically disordered proteins (IDPs) containing phenylalanyl-glycyl (FG)-rich repeats line the pore and interact with TFs. However, the reason that transport can be both fast and specific remains undetermined, through lack of atomic-scale information on the behavior of FGs and their interaction with TFs. We used nuclear magnetic resonance spectroscopy to address these issues. We show that FG repeats are highly dynamic IDPs, stabilized by the cellular environment. Fast transport of TFs is supported because the rapid motion of FG motifs allows them to exchange on and off TFs extremely quickly through transient interactions. Because TFs uniquely carry multiple pockets for FG repeats, only they can form the many frequent interactions needed for specific passage between FG repeats to cross the NPC.

Controlling lipid micelle stability using oligonucleotide headgroups.

Lipid-based micelles provide an attractive option for therapeutic and diagnostic applications because of their small size (<20 nm) and ability to self-assemble and improve the solubility of both hydrophobic drugs and dyes. Their use, however, has been challenged by the fact that these particles are inherently unstable in serum becaue of interactions with protein components, which drives the micelle equilibrium to the monomeric state. We have engineered serum stabilized micelles using short quadruplex forming oligonucleotide extensions as the lipid headgroup. Quadruplex formation on the surface of the particles, confirmed by (1)H NMR, results in slight distortion of the otherwise spherical micelles and renders them resistant to disassembly by serum proteins for >24 h. Using antisense oligonucleotides we demonstrated that disruption of the quadruplex leads to micelle destabilization and cargo release. The ability to use oligonucleotide interactions to control lipid particle stability represents a new approach in the design of programmed nanoscale devices.

Curcumin modulates the self-assembly of the islet amyloid polypeptide by disassembling α-helix.

Understanding how small molecules affect amyloid formation is of major biomedical and pharmaceutical importance due to the association of amyloid with incurable diseases including Alzheimer's, Parkinson's, and type II diabetes. Using solution state (1)H NMR, we demonstrate that curcumin, a planar biphenolic compound found in the Indian spice turmeric, delays the self-assembly of islet amyloid polypeptide to NMR-invisible assemblies. Accompanying circular dichroism studies show that curcumin disassembles α-helix in maturing assemblies of IAPP. The amount of α-helix disassembled correlates with predicted and experimentally determined helical content of IAPP obtained by others. Taken together, these results indicate that curcumin modulates IAPP self-assembly by unfolding α-helix on pathway to amyloid. The implications of this work in the elucidation of the mechanism for amyloid formation by IAPP in the presence of curcumin are discussed.

Helix-dipole effects in peptide self-assembly to amyloid.

The formation of amyloid fibrils is associated with incurable diseases including Alzheimer's, Parkinson's, and type 2 diabetes. Important mechanistic details of the self-assembly are unknown partly because of the absence of a clear structural characterization of intermediates. There is experimental evidence, however, for α-helical intermediates that has come primarily from circular dichroism spectroscopy. Here, we strengthen the evidence for helical intermediates by demonstrating helix-dipole effects in the early events of self-assembly. Previously, we showed that capped peptides containing the part of the islet amyloid polypeptide that may be responsible for the initial intermolecular contacts (Acetyl-R(11)LANFLVHSSNNFGA(25)-NH(2) and Acetyl-R(11)LANFLVHSGNNFGA(25)-NH(2) which contains the S20G mutation associated with early onset type 2 diabetes) self-assemble via helical intermediates [Liu et al. (2010) J. Am. Chem. Soc.132, 18223-18232]. We demonstrate here that when the peptides are uncapped, they do not self-assemble as indicated primarily by circular dichroism and nuclear magnetic resonance data. Self-assembly is restored when the charge on α-NH(3)(+) of Arg11 is eliminated but not when the charge on α-COO(-) of Ala25 is removed, consistent with the helicity of the peptides skewed toward the N-terminus. Our results strengthen the hypothesis that α-helical intermediates are on pathway to amyloid formation and indicate that the helix dipole is an attractive target for inhibiting the formation of α-helical assemblies.