Phase separation of intrinsically disordered FG-Nups is driven by highly dynamic FG motifs.

The intrinsically disordered FG-Nups in the central channel of the nuclear pore complex (NPC) form a selective permeability barrier, allowing small molecules to traverse by passive diffusion, while large molecules can only translocate with the help of nuclear transport receptors. The exact phase state of the permeability barrier remains elusive. In vitro experiments have shown that some FG-Nups can undergo phase separation into condensates that display NPC-like permeability barrier properties. Here, we use molecular dynamics simulations at amino acid resolution to study the phase separation characteristics of each of the disordered FG-Nups of the yeast NPC. We find that GLFG-Nups undergo phase separation and reveal that the FG motifs act as highly dynamic hydrophobic stickers that are essential for the formation of FG-Nup condensates featuring droplet-spanning percolated networks. Additionally, we study phase separation in an FG-Nup mixture that resembles the NPC stoichiometry and observe that an NPC condensate is formed containing multiple GLFG-Nups. We find that the phase separation of this NPC condensate is also driven by FG-FG interactions, similar to the homotypic FG-Nup condensates. Based on the observed phase separation behavior, the different FG-Nups of the yeast NPC can be divided into two classes: The FG-Nups (mostly GLFG-type) located in the central channel of the NPC form a highly dynamic percolated network formed by many short-lived FG-FG interactions, while the peripheral FG-Nups (mostly FxFG-type) at the entry and exit of the NPC channel likely form an entropic brush.

Exploring Intra- and Inter-Regional Interactions in the IDP α-Synuclein Using smFRET and MD Simulations.

Theoretical concepts from polymer physics are often used to describe intrinsically disordered proteins (IDPs). However, amino acid interactions within and between regions of the protein can lead to deviations from typical polymer scaling behavior and even to short-lived secondary structures. To investigate the key interactions in the dynamic IDP α-synuclein (αS) at the amino acid level, we conducted single-molecule fluorescence resonance energy transfer (smFRET) experiments and coarse-grained molecular dynamics (CG-MD) simulations. We find excellent agreement between experiments and simulations. Our results show that a physiological salt solution is a good solvent for αS and that the protein is highly dynamic throughout its entire chain, with local intra- and inter-regional interactions leading to deviations from global scaling. Specifically, we observe expansion in the C-terminal region, compaction in the NAC region, and a slightly smaller distance between the C- and N-termini than expected. Our simulations indicate that the compaction in the NAC region results from hydrophobic aliphatic contacts, mostly between valine and alanine residues, and cation-π interactions between lysine and tyrosine. In addition, hydrogen bonds also seem to contribute to the compaction of the NAC region. The expansion of the C-terminal region is due to intraregional electrostatic repulsion and increased chain stiffness from several prolines. Overall, our study demonstrates the effectiveness of combining smFRET experiments with CG-MD simulations to investigate the key interactions in highly dynamic IDPs at the amino acid level.

Ultra-low-density digitally architected carbon with a strutted tube-in-tube structure.

Porous materials with engineered stretching-dominated lattice designs, which offer attractive mechanical properties with ultra-light weight and large surface area for wide-ranging applications, have recently achieved near-ideal linear scaling between stiffness and density. Here, rather than optimizing the microlattice topology, we explore a different approach to strengthen low-density structural materials by designing tube-in-tube beam structures. We develop a process to transform fully dense, three-dimensional printed polymeric beams into graphitic carbon hollow tube-in-tube sandwich morphologies, where, similar to grass stems, the inner and outer tubes are connected through a network of struts. Compression tests and computational modelling show that this change in beam morphology dramatically slows down the decrease in stiffness with decreasing density. In situ pillar compression experiments further demonstrate large deformation recovery after 30-50% compression and high specific damping merit index. Our strutted tube-in-tube design opens up the space and realizes highly desirable high modulus-low density and high modulus-high damping material structures.

Metachronal patterns by magnetically-programmable artificial cilia surfaces for low Reynolds number fluid transport and mixing.

Motile cilia can produce net fluid flows at low Reynolds number because of their asymmetric motion and metachrony of collective beating. Mimicking this with artificial cilia can find application in microfluidic devices for fluid transport and mixing. Here, we study the metachronal beating of nonidentical, magnetically-programmed artificial cilia whose individual non-reciprocal motion and collective metachronal beating pattern can be independently controlled. We use a finite element method that accounts for magnetic forces, cilia deformation and fluid flow in a fully coupled manner. Mimicking biological cilia, we study magnetic cilia subject to a full range of metachronal driving patterns, including antiplectic, symplectic, laeoplectic and diaplectic waves. We analyse the induced primary flow, secondary flow and mixing rate as a function of the phase lag between cilia and explore the underlying physical mechanism. Our results show that shielding effects between neighboring cilia lead to a primary flow that is larger for antiplectic than for symplectic metachronal waves. The secondary flow can be fully explained by the propagation direction of the metachronal wave. Finally, we show that the mixing rate can be strongly enhanced by laeoplectic and diaplectic metachrony resulting in large velocity gradients and vortex-like flow patterns.

Two Sides of the Same Coin: Emergence of Foldamers and Self-Replicators from Dynamic Combinatorial Libraries.

The ability of molecules and systems to make copies of themselves and the ability of molecules to fold into stable, well-defined three-dimensional conformations are of considerable importance in the formation and persistence of life. The question of how, during the emergence of life, oligomerization reactions become selective and channel these reactions toward a small number of specific products remains largely unanswered. Herein, we demonstrate a fully synthetic chemical system where structurally complex foldamers and self-replicating assemblies emerge spontaneously and with high selectivity from pools of oligomers as a result of forming noncovalent interactions. Whether foldamers or replicators form depends on remarkably small differences in building block structures and composition and experimental conditions. We also observed the dynamic transformation of a foldamer into a replicator. These results show that the structural requirements/design criteria for building blocks that lead to foldamers are similar to those that lead to replicators. What determines whether folding or replication takes place is not necessarily the type of noncovalent interaction, but only whether they occur intra- or intermolecularly. This work brings together, for the first time, the fields of replicator and foldamer chemistry.

Physics of the Nuclear Pore Complex: Theory, Modeling and Experiment.

The hallmark of eukaryotic cells is the nucleus that contains the genome, enclosed by a physical barrier known as the nuclear envelope (NE). On the one hand, this compartmentalization endows the eukaryotic cells with high regulatory complexity and flexibility. On the other hand, it poses a tremendous logistic and energetic problem of transporting millions of molecules per second across the nuclear envelope, to facilitate their biological function in all compartments of the cell. Therefore, eukaryotes have evolved a molecular "nanomachine" known as the Nuclear Pore Complex (NPC). Embedded in the nuclear envelope, NPCs control and regulate all the bi-directional transport between the cell nucleus and the cytoplasm. NPCs combine high molecular specificity of transport with high throughput and speed, and are highly robust with respect to molecular noise and structural perturbations. Remarkably, the functional mechanisms of NPC transport are highly conserved among eukaryotes, from yeast to humans, despite significant differences in the molecular components among various species. The NPC is the largest macromolecular complex in the cell. Yet, despite its significant complexity, it has become clear that its principles of operation can be largely understood based on fundamental physical concepts, as have emerged from a combination of experimental methods of molecular cell biology, biophysics, nanoscience and theoretical and computational modeling. Indeed, many aspects of NPC function can be recapitulated in artificial mimics with a drastically reduced complexity compared to biological pores. We review the current physical understanding of the NPC architecture and function, with the focus on the critical analysis of experimental studies in cells and artificial NPC mimics through the lens of theoretical and computational models. We also discuss the connections between the emerging concepts of NPC operation and other areas of biophysics and bionanotechnology.

Phase Separation of Toxic Dipeptide Repeat Proteins Related to C9orf72 ALS/FTD.

The expansion mutation in the C9orf72 gene is the most common known genetic cause for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This mutation can produce five dipeptide repeat proteins (DPRs), of which three are known to be toxic: poly-PR, poly-GR, and poly-GA. The toxicity of poly-GA is attributed to its aggregation in the cytoplasm, whereas for poly-PR and poly-GR, several toxicity pathways have been proposed. The toxicity of the DPRs has been shown to depend on their length, but the underlying molecular mechanism of this length dependence is not well understood. To address the possible role of phase separation in DPR toxicity, a one-bead-per-amino-acid (1BPA) coarse-grained molecular dynamics model is used to study the single-molecule and phase-separation properties of the DPRs. We find a strong dependence of the phase-separation behavior on both DPR length and concentration, with longer DPRs having a higher propensity to phase separate and form condensed phases with higher concentrations. The critical lengths required for phase separation (25 for poly-PR and 50 for poly-GA) are comparable to the toxicity threshold limit of 30 repeats found for the expansion mutation in patient cells, suggesting that phase separation could play an important role in DPR toxicity.

Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements.

Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration (RG ), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance (RE ) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values RG and RE For chemically denatured proteins we obtain mutual consistency in our inferences based on RG and RE , whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between RE and RG that is amplified in the absence of denaturants. Therefore, joint assessments of RG and RE combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles.

Travelling waves on photo-switchable patterned liquid crystal polymer films directed by rotating polarized light.

Nature employs travelling waves to generate propulsion of fluids, cells and organisms. This has inspired the development of responsive material systems based on different external triggers. Especially light-actuation is suitable because of its remote control and scalability, but often complex, moving light sources are required. Here, we developed a method that only requires flood exposure by rotating the linear polarization of light to generate propagating surface waves on azobenzene-modified liquid crystalline polymer films. We built a photomechanical computational model that accounts for the attenuation of polarized light and trans-to-cis isomerization of azobenzene. A non-uniform in-plane distribution of the liquid crystal molecules allows for the generation of travelling surface waves whose amplitude, speed and direction can be controlled through the intensity, rotation direction and rotation speed of the linear polarization of a light source. Our method opens new avenues for motion control based on light-responsive topographical transformations for application in microfluidic lab-on-chip systems and soft robotics.

Pillars or Pancakes? Self-Cleaning Surfaces without Coating.

Surfaces that stay clean when immersed in water are important for an enormous range of applications from ships and buildings to marine, medical, and other equipment. Up until now the main strategy for designing self-cleaning surfaces has been to combine hydrophilic/hydrophobic coatings with a high aspect ratio structuring (typically micron scale pillars) to trap a (semi)static water/air layer for drag and adhesion reduction. However, such coating and structuring can distort optical properties and get damaged in harsh environments, and contamination, i.e., particles, oil droplets, and biofouling, can get trapped and aggregate in the structure. Here we present a radically different strategy for self-cleaning surface design: We show that a surface can be made self-cleaning by structuring with a pattern of very low aspect ratio pillars ("pancakes"). Now the water is not trapped. It can flow freely around the pancakes thus creating a dynamic water layer. We have applied the new pancake design to sapphire windows and made the first surfaces that are self-cleaning through structuring alone without the application of any coating. An offshore installation has now been running continuously with structured windows for more than one year. The previous uptime for unstructured windows was 7 days.

The Effect of FG-Nup Phosphorylation on NPC Selectivity: A One-Bead-Per-Amino-Acid Molecular Dynamics Study.

Nuclear pore complexes (NPCs) are large protein complexes embedded in the nuclear envelope separating the cytoplasm from the nucleoplasm in eukaryotic cells. They function as selective gates for the transport of molecules in and out of the nucleus. The inner wall of the NPC is coated with intrinsically disordered proteins rich in phenylalanine-glycine repeats (FG-repeats), which are responsible for the intriguing selectivity of NPCs. The phosphorylation state of the FG-Nups is controlled by kinases and phosphatases. In the current study, we extended our one-bead-per-amino-acid (1BPA) model for intrinsically disordered proteins to account for phosphorylation. With this, we performed molecular dynamics simulations to probe the effect of phosphorylation on the Stokes radius of isolated FG-Nups, and on the structure and transport properties of the NPC. Our results indicate that phosphorylation causes a reduced attraction between the residues, leading to an extension of the FG-Nups and the formation of a significantly less dense FG-network inside the NPC. Furthermore, our simulations show that upon phosphorylation, the transport rate of inert molecules increases, while that of nuclear transport receptors decreases, which can be rationalized in terms of modified hydrophobic, electrostatic, and steric interactions. Altogether, our models provide a molecular framework to explain how extensive phosphorylation of FG-Nups decreases the selectivity of the NPC.

Mechanisms of influenza viral membrane fusion.

Influenza viral particles are enveloped by a lipid bilayer. A major step in infection is fusion of the viral and host cellular membranes, a process with large kinetic barriers. Influenza membrane fusion is catalyzed by hemagglutinin (HA), a class I viral fusion protein activated by low pH. The exact nature of the HA conformational changes that deliver the energy required for fusion remains poorly understood. This review summarizes our current knowledge of HA structure and dynamics, describes recent single-particle experiments and modeling studies, and discusses their role in understanding how multiple HAs mediate fusion. These approaches provide a mechanistic picture in which HAs independently and stochastically insert into the target membrane, forming a cluster of HAs that is collectively able to overcome the barrier to membrane fusion. The new experimental and modeling approaches described in this review hold promise for a more complete understanding of other viral fusion systems and the protein systems responsible for cellular fusion.

Topographical changes in photo-responsive liquid crystal films: a computational analysis.

Switchable materials in response to external stimuli serve as building blocks to construct microscale functionalized actuators and sensors. Azobenzene-modified liquid crystal (LC) polymeric networks, that combine liquid crystalline orientational order and elasticity, reversibly undergo conformational changes powered by light. We present a computational framework to describe photo-induced topographical transformations of azobenzene-modified LC glassy polymer coatings. A nonlinear light penetration model is combined with an opto-mechanical constitutive relation to simulate various ordered and corrugated topographical textures resulting from aligned or randomly distributed LC molecule orientations. Our results shed light on the fundamental physical mechanisms of light-triggered surface undulations and can be used as guidelines to optimize surface modulation and roughness in emerging fields that involve haptics interfacing, friction control and wetting manipulation.

Reverse switching of surface roughness in a self-organized polydomain liquid crystal coating.

In this work we propose randomly ordered polydomain nematic liquid crystal polymer networks to reversibly generate notable jagged relief patterns at a polymer coating surface by light illumination. The domain size is controlled by the addition of traces of partly insoluble fluorinated acrylate. The photoresponse of the coating is induced by a small amount of copolymerized azobenzene monomers. Upon exposure to UV light, azobenzene undergoes trans to cis isomerization, resulting in a change in molecular order and packing within each domain. The extent of this effect and its directionality depends on the domain orientation. Localized to domain level, this morphological change forms large 3D spikes at the surface with a modulation amplitude of more than 20% of the initial thickness. The process is reversible; the surface topographical patterns erase within 10 s by stopping the light exposure. A finite element model is applied to simulate the surface topography changes of the polydomain coating. The simulations describe the formation of the topographic features in terms of light absorption and isomerization process as a function of the director orientation. The random director distribution leads to surface structures which were found to be in close agreement with the ones measured by interference microscopy. The effect of domain size on surface roughness and depth modulation was explored and related to the internal mechanical constraints. The use of nematic liquid crystal polydomains confined in a polymer network largely simplifies the fabrication of smart coatings with a prominent triggered topographic response.

Enhanced Deformation of Azobenzene-Modified Liquid Crystal Polymers under Dual Wavelength Exposure: A Photophysical Model.

Azobenzene-embedded liquid crystal polymers can undergo mechanical deformation in response to ultraviolet (UV) light. The natural rodlike trans state azobenzene absorbs UV light and isomerizes to a bentlike cis state, which disturbs the order of the polymer network, leading to an anisotropic deformation. The current consensus is that the magnitude of the photoinduced deformation is related to the statistical building up of molecules in the cis state. However, a recent experimental study [Liu and Broer, Nat. Commun. 6 8334 (2015).NCAOBW2041-172310.1038/ncomms9334] shows that a drastic (fourfold) increase of the photoinduced deformation can be generated by exposing the samples simultaneously to 365 nm (UV) and 455 nm (visible) light. To elucidate the physical mechanism that drives this increase, we develop a two-light attenuation model and an optomechanical constitutive relation that not only accounts for the statistical accumulation of cis azobenzenes, but also for the dynamic trans-cis-trans oscillatory isomerization process. Our experimentally calibrated model predicts that the optimal single-wavelength exposure is 395 nm light, a pronounced shift towards the visible spectrum. In addition, we identify a range of optimal combinations of two-wavelength lights that generate a favorable response for a given amount of injected energy. Our model provides mechanistic insight into the different (multi)wavelength exposures used in experiments and, at the same time, opens new avenues towards enhanced, multiwavelength optomechanical behavior.

Two fundamental mechanisms govern the stiffening of cross-linked networks.

Biopolymer networks, such as those constituting the cytoskeleton of a cell or biological tissue, exhibit a nonlinear strain-stiffening behavior when subjected to large deformations. Interestingly, rheological experiments on various in vitro biopolymer networks have shown similar strain-stiffening trends regardless of the differences in their microstructure or constituents, suggesting a universal stiffening mechanism. In this article, we use computer simulations of a random network comprised of cross-linked biopolymer-like fibers to substantiate the notion that this universality lies in the existence of two fundamental stiffening mechanisms. After showing that the large strain response is accompanied by the development of a stress path, i.e., a percolating path of axially stressed fibers and cross-links, we demonstrate that the strain stiffening can be caused by two distinctly different mechanisms: 1) the pulling out of stress-path undulations; and 2) reorientation of the stress path. The former mechanism is bending-dominated and can be recognized by a power-law dependence with exponent 3/2 of the shear modulus on stress, whereas the latter mechanism is stretching-dominated and characterized by a power-law exponent 1/2. We demonstrate how material properties of the constituents, as well as the network microstructure, can affect the transition between the two stiffening mechanisms and, as such, control the dominant power-law scaling behavior.

Probing the disordered domain of the nuclear pore complex through coarse-grained molecular dynamics simulations.

The distribution of disordered proteins (FG-nups) that line the transport channel of the nuclear pore complex (NPC) is investigated by means of coarse-grained molecular dynamics simulations. A one-bead-per-amino-acid model is presented that accounts for the hydrophobic/hydrophilic and electrostatic interactions between different amino acids, polarity of the solvent, and screening of free ions. The results indicate that the interaction of the FG-nups forms a high-density, doughnut-like distribution inside the NPC, which is rich in FG-repeats. We show that the obtained distribution is encoded in the amino-acid sequence of the FG-nups and is driven by both electrostatic and hydrophobic interactions. To explore the relation between structure and function, we have systematically removed different combinations of FG-nups from the pore to simulate inviable and viable NPCs that were previously studied experimentally. The obtained density distributions show that the maximum density of the FG-nups inside the pore does not exceed 185 mg/mL in the inviable NPCs, whereas for the wild-type and viable NPCs, this value increases to 300 mg/mL. Interestingly, this maximum density is not correlated to the total mass of the FG-nups, but depends sensitively on the specific combination of essential Nups located in the central plane of the NPC.

C9orf72 polyPR directly binds to various nuclear transport components.

The disruption of nucleocytoplasmic transport (NCT) is an important mechanism in neurodegenerative diseases. In the case of C9orf72-ALS, trafficking of macromolecules through the nuclear pore complex (NPC) might get frustrated by the binding of C9orf72-translated arginine-containing dipeptide repeat proteins (R-DPRs) to the Kapß family of nuclear transport receptors. Besides Kapßs, several other types of transport components have been linked to NCT impairments in R-DPR-expressed cells, but the molecular origin of these observations has not been clarified. Here, we adopt a coarse-grained molecular dynamics model at amino acid resolution to study the direct interaction between polyPR, the most toxic DPR, and various nuclear transport components to elucidate the binding mechanisms and provide a complete picture of potential polyPR-mediated NCT defects. We found polyPR to directly bind to several isoforms of the Impα family, CAS (the specific exporter of Impα) and RanGAP. We observe no binding between polyPR and Ran. Longer polyPRs at lower salt concentrations also make contact with RanGEF and NTF2. Analyzing the polyPR contact sites on the transport components reveals that polyPR potentially interferes with RanGTP/RanGDP binding, with nuclear localization signal (NLS)-containing cargoes (cargo-NLS) binding to Impα, with cargo-NLS release from Impα, and with Impα export from the nucleus. The abundance of polyPR-binding sites on multiple transport components combined with the inherent polyPR length dependence makes direct polyPR interference of NCT a potential mechanistic pathway of C9orf72 toxicity.

Tertiary structure and conformational dynamics of the anti-amyloidogenic chaperone DNAJB6b at atomistic resolution.

DNAJB6b is a molecular chaperone of the heat shock protein network, shown to play a crucial role in preventing aggregation of several disease-related intrinsically disordered proteins. Using homology modeling and microsecond-long all-atom molecular dynamics (MD) simulations, we show that monomeric DNAJB6b is a transiently interconverting protein cycling between three states: a closed state, an open state (both abundant), and a less abundant extended state. Interestingly, the reported regulatory autoinhibitory anchor between helix V in the G/F1 region and helices II/III of the J-domain, which obstructs the access of Hsp70 to the J-domain remains present in all three states. This possibly suggests a mechanistically intriguing regulation in which DNAJB6b only becomes exposed when loaded with substrates that require Hsp70 processing. Our MD results of DNAJB6b carrying mutations in the G/F1 region that are linked to limb-girdle muscular dystrophy type D1 (LGMDD1) show that this G/F1 region becomes highly dynamic, pointing towards a spontaneous release of the autoinhibitory helix V from helices II/III. This would increase the probability of non-functional Hsp70 interactions to DNAJB6b without substrates. Our cellular data indeed confirm that non-substrate loaded LGMDD1 mutants have aberrant interactions with Hsp70.

Diameter dependence of transport through nuclear pore complex mimics studied using optical nanopores.

The nuclear pore complex (NPC) regulates the selective transport of large biomolecules through the nuclear envelope. As a model system for nuclear transport, we construct NPC mimics by functionalizing the pore walls of freestanding palladium zero-mode waveguides with the FG-nucleoporin Nsp1. This approach enables the measurement of single-molecule translocations through individual pores using optical detection. We probe the selectivity of Nsp1-coated pores by quantitatively comparing the translocation rates of the nuclear transport receptor Kap95 to the inert probe BSA over a wide range of pore sizes from 35 nm to 160 nm. Pores below 55 ± 5 nm show significant selectivity that gradually decreases for larger pores. This finding is corroborated by coarse-grained molecular dynamics simulations of the Nsp1 mesh within the pore, which suggest that leakage of BSA occurs by diffusion through transient openings within the dynamic mesh. Furthermore, we experimentally observe a modulation of the BSA permeation when varying the concentration of Kap95. The results demonstrate the potential of single-molecule fluorescence measurements on biomimetic NPCs to elucidate the principles of nuclear transport.