Protein-membrane interactions: sensing and generating curvature.

Biological membranes are integral cellular structures that can be curved into various geometries. These curved structures are abundant in cells as they are essential for various physiological processes. However, curved membranes are inherently unstable, especially on nanometer length scales. To stabilize curved membranes, cells can utilize proteins that sense and generate membrane curvature. In this review, we summarize recent research that has advanced our understanding of interactions between proteins and curved membrane surfaces, as well as work that has expanded our ability to study curvature sensing and generation. Additionally, we look at specific examples of cellular processes that require membrane curvature, such as neurotransmission, clathrin-mediated endocytosis (CME), and organelle biogenesis.

Mechanistic divergences of endocytic clathrin-coated vesicle formation in mammals, yeasts and plants.

Clathrin-coated vesicles (CCVs), generated by clathrin-mediated endocytosis (CME), are essential eukaryotic trafficking organelles that transport extracellular and plasma membrane-bound materials into the cell. In this Review, we explore mechanisms of CME in mammals, yeasts and plants, and highlight recent advances in the characterization of endocytosis in plants. Plants separated from mammals and yeast over 1.5 billion years ago, and plant cells have distinct biophysical parameters that can influence CME, such as extreme turgor pressure. Plants can therefore provide a wider perspective on fundamental processes in eukaryotic cells. We compare key mechanisms that drive CCV formation and explore what these mechanisms might reveal about the core principles of endocytosis across the tree of life. Fascinatingly, CME in plants appears to more closely resemble that in mammalian cells than that in yeasts, despite plants being evolutionarily further from mammals than yeast. Endocytic initiation appears to be highly conserved across these three systems, requiring similar protein domains and regulatory processes. Clathrin coat proteins and their honeycomb lattice structures are also highly conserved. However, major differences are found in membrane-bending mechanisms. Unlike in mammals or yeast, plant endocytosis occurs independently of actin, highlighting that mechanistic assumptions about CME across different systems should be made with caution.

The vaccinia chondroitin sulfate binding protein drives host membrane curvature to facilitate fusion.

Cellular attachment of viruses determines their cell tropism and species specificity. For entry, vaccinia, the prototypic poxvirus, relies on four binding proteins and an eleven-protein entry fusion complex. The contribution of the individual virus binding proteins to virion binding orientation and membrane fusion is unclear. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards the virus fusion machinery to facilitate fusion. Using a membrane-bleb model system together with super-resolution and electron microscopy we find that side-bound vaccinia virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins reveals that three of four contribute to binding orientation, amongst which the chondroitin sulfate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection. Our results show that viral binding proteins are active participants in successful virus membrane fusion and illustrate the importance of virus protein architecture for successful infection.

Cracking the DNA Code for V(D)J Recombination.

To initiate V(D)J recombination for generating the adaptive immune response of vertebrates, RAG1/2 recombinase cleaves DNA at a pair of recombination signal sequences, the 12- and 23-RSS. We have determined crystal and cryo-EM structures of RAG1/2 with DNA in the pre-reaction and hairpin-forming complexes up to 2.75 Å resolution. Both protein and DNA exhibit structural plasticity and undergo dramatic conformational changes. Coding-flank DNAs extensively rotate, shift, and deform for nicking and hairpin formation. Two intertwined RAG1 subunits crisscross four times between the asymmetric pair of severely bent 12/23-RSS DNAs. Location-sensitive bending of 60° and 150° in 12- and 23-RSS spacers, respectively, must occur for RAG1/2 to capture the nonamers and pair the heptamers for symmetric double-strand breakage. DNA pairing is thus sequence-context dependent and structure specific, which partly explains the "beyond 12/23" restriction. Finally, catalysis in crystallo reveals the process of DNA hairpin formation and its stabilization by interleaved base stacking.

Biophysical aspects of migrasome organelle formation and their diverse cellular functions.

The transient cellular organelles known as migrasomes, which form during cell migration along retraction fibers, have emerged as a crutial factor in various fundamental cellular processes and pathologies. These membrane vesicles originate from local membrane swellings, encapsulate specific cytoplasmic content, and are eventually released to the extracellular environment or taken up by recipient cells. Migrasome biogenesis entails a sequential membrane remodeling process involving a complex interplay between various molecular factors such as tetraspanin proteins, and mechanical properties like membrane tension and bending rigidity. In this review, we summarize recent studies exploring the mechanism of migrasome formation. We emphasize how physical forces, together with molecular factors, shape migrasome biogenesis, and detail the involvement of migrasomes in various cellular processes and pathologies. A comprehensive understanding of the exact mechanism underlying migrasome formation and the identification of key molecules involved hold promise for advancing their therapeutic and diagnostic applications.

Direct mapping of bending and torsional dynamics in individual nanostructures.

Investigating coherent acoustic vibrations in nanostructured materials provides fundamental insights into optomechanical responses and microscopic energy flow. Extensive measurements of vibrational dynamics have been performed for a wide variety of nanoparticles and nanoparticle assemblies. However, virtually all of them show that only the dilation modes are launched after laser excitations, and the acoustic bending and torsional motions, which are commonly observed in photoexcited chemical bonds, are absent. Unambiguous identification and refined characterization of these "missing" modes have been a long-standing issue. In this report, we investigated the acoustic vibrational dynamics of individual Au nanoprisms on free-standing graphene substrates using an ultrafast high-sensitivity dark-field imaging approach in four-dimensional transmission electron microscopy. Following optical excitations, we observed low-frequency multiple-mode oscillations and higher superposition amplitudes at nanoprism corners and edges on the subnanoparticle level. In combination with finite-element simulations, we determined that these vibrational modes correspond to out-of-plane bending and torsional motions, superimposed by an overall tilting effect of the nanoprisms. The launch and relaxation processes of these modes are highly pertinent to substrate effects and nanoparticle geometries. These findings contribute to the fundamental understanding about acoustic dynamics of individual nanostructures and their interaction with substrates.

Role of nucleobase-specific interactions in the binding and bending of DNA by human male sex determination factor SRY.

Y-chromosome-encoded master transcription factor SRY functions in the embryogenesis of therian mammals to initiate male development. Through interactions of its conserved high-mobility group box within a widened DNA minor groove, SRY and related Sox factors induce sharp bends at specific DNA target sites. Here, we present the crystal structure of the SRY high-mobility group domain bound to a DNA site containing consensus element 5'-ATTGTT. The structure contains three complexes in the asymmetric unit; in each complex, SRY forms 10 hydrogen bonds with minor-groove base atoms in 5'-CATTGT/ACAATG-3', shifting the recognition sequence by one base pair (italics). These nucleobase interactions involve conserved residues Arg7, Asn10, and Tyr74 on one side of intercalated Ile13 (the cantilever) and Arg20, Asn32, and Ser36 on the other. Unlike the less-bent NMR structure, DNA bend angles (69-84°) of the distinct box-DNA complexes are similar to those observed in homologous Sox domain-DNA structures. Electrophoretic studies indicate that respective substitutions of Asn32, Ser36, or Tyr74 by Ala exhibit slightly attenuated specific DNA-binding affinity and bend angles (70-73°) relative to WT (79°). By contrast, respective substitutions of Arg7, Asn10, or Arg20 by Ala markedly impaired DNA-binding affinity in association with much smaller DNA bend angles (53-65°). In a rodent cell-based model of the embryonic gonadal ridge, full-length SRY variants bearing these respective Ala substitutions exhibited significantly decreased transcriptional activation of SRY's principal target gene (Sox9). Together, our findings suggest that nucleobase-specific hydrogen bonds by SRY are critical for specific DNA binding, bending, and transcriptional activation.

Bacteriophage lambda site-specific recombination.

The site-specific recombination pathway of bacteriophage λ encompasses isoenergetic but highly directional and tightly regulated integrative and excisive reactions that integrate and excise the vial chromosome into and out of the bacterial chromosome. The reactions require 240 bp of phage DNA and 21 bp of bacterial DNA comprising 16 protein binding sites that are differentially used in each pathway by the phage-encoded Int and Xis proteins and the host-encoded integration host factor and factor for inversion stimulation proteins. Structures of higher-order protein-DNA complexes of the four-way Holliday junction recombination intermediates provided clarifying insights into the mechanisms, directionality, and regulation of these two pathways, which are tightly linked to the physiology of the bacterial host cell. Here we review our current understanding of the mechanisms responsible for regulating and executing λ site-specific recombination, with an emphasis on key studies completed over the last decade.

Admp regulates tail bending by controlling ventral epidermal cell polarity via phosphorylated myosin localization in Ciona.

Ventral tail bending, which is transient but pronounced, is found in many chordate embryos and constitutes an interesting model of how tissue interactions control embryo shape. Here, we identify one key upstream regulator of ventral tail bending in embryos of the ascidian Ciona. We show that during the early tailbud stages, ventral epidermal cells exhibit a boat-shaped morphology (boat cell) with a narrow apical surface where phosphorylated myosin light chain (pMLC) accumulates. We further show that interfering with the function of the BMP ligand Admp led to pMLC localizing to the basal instead of the apical side of ventral epidermal cells and a reduced number of boat cells. Finally, we show that cutting ventral epidermal midline cells at their apex using an ultraviolet laser relaxed ventral tail bending. Based on these results, we propose a previously unreported function for Admp in localizing pMLC to the apical side of ventral epidermal cells, which causes the tail to bend ventrally by resisting antero-posterior notochord extension at the ventral side of the tail.

Establishing finite element model credibility of a pedicle screw system under compression-bending: An end-to-end example of the ASME V&V 40 standard.

Computational modeling and simulation (CM&S) is a key tool in medical device design, development, and regulatory approval. For example, finite element analysis (FEA) is widely used to understand the mechanical integrity and durability of orthopaedic implants. The ASME V&V 40 standard and supporting FDA guidance provide a framework for establishing model credibility, enabling deeper reliance on CM&S throughout the total product lifecycle. Examples of how to apply the principles outlined in the ASME V&V 40 standard are important to facilitating greater adoption by the medical device community, but few published examples are available that demonstrate best practices. Therefore, this paper outlines an end-to-end (E2E) example of the ASME V&V 40 standard applied to an orthopaedic implant. The objective of this study was to illustrate how to establish the credibility of a computational model intended for use as part of regulatory evaluation. In particular, this study focused on whether a design change to a spinal pedicle screw construct (specifically, the addition of a cannulation to an existing non-cannulated pedicle screw) would compromise the rod-screw construct mechanical performance. This question of interest (?OI) was addressed by establishing model credibility requirements according to the ASME V&V 40 standard. Experimental testing to support model validation was performed using spinal rods and non-cannulated pedicle screw constructs made with medical grade titanium (Ti-6Al-4V ELI). FEA replicating the experimental tests was performed by three independent modelers and validated through comparisons of common mechanical properties such as stiffness and yield force. The validated model was then used to simulate F1717 compression-bending testing on the new cannulated pedicle screw design to answer the ?OI, without performing any additional experimental testing. This E2E example provides a realistic scenario for the application of the ASME V&V 40 standard to orthopedic medical device applications.

The underlying mechanical properties of membranes tune their ability to fuse.

Membrane fusion is a ubiquitous process associated with a multitude of biological events. Although it has long been appreciated that membrane mechanics plays an important role in membrane fusion, the molecular interplay between mechanics and fusion has remained elusive. For example, although different lipids modulate membrane mechanics differently, depending on their composition, molar ratio, and complex interactions, differing lipid compositions may lead to similar mechanical properties. This raises the question of whether (i) the specific lipid composition or (ii) the average mesoscale mechanics of membranes acts as the determining factor for cellular function. Furthermore, little is known about the potential consequences of fusion on membrane disruption. Here, we use a combination of confocal microscopy, time-resolved imaging, and electroporation to shed light onto the underlying mechanical properties of membranes that regulate membrane fusion. Fusion efficiency follows a nearly universal behavior that depends on membrane fluidity parameters, such as membrane viscosity and bending rigidity, rather than on specific lipid composition. This helps explaining why the charged and fluid membranes of the inner leaflet of the plasma membrane are more fusogenic than their outer counterparts. Importantly, we show that physiological levels of cholesterol, a key component of biological membranes, has a mild effect on fusion but significantly enhances membrane mechanical stability against pore formation, suggesting that its high cellular levels buffer the membrane against disruption. The ability of membranes to efficiently fuse while preserving their integrity may have given evolutionary advantages to cells by enabling their function while preserving membrane stability.

Limiting etioplast gene expression induces apical hook twisting during skotomorphogenesis of Arabidopsis seedlings.

When covered by a layer of soil, seedling development follows a dark-specific program (skotomorphogenesis). In the dark, seedlings consist of small, non-green cotyledons, a long hypocotyl, and an apical hook to protect meristematic cells. We recently highlighted the role played by mitochondria in the high energy-consuming reprogramming of Arabidopsis skotomorphogenesis. Here, the role played by plastids, another energy-supplying organelle, in skotomorphogenesis is investigated. This study was conducted in dark conditions to exclude light signals so as to better focus on those produced by plastids. It was found that limitation of plastid gene expression (PGE) induced an exaggerated apical hook bending. Inhibition of PGE was obtained at the levels of transcription and translation using the antibiotics rifampicin (RIF) and spectinomycin, respectively, as well as plastid RPOTp RNA polymerase mutants. RIF-treated seedlings also showed expression induction of marker nuclear genes for mitochondrial stress, perturbation of mitochondrial metabolism, increased ROS levels, and an augmented capacity of oxygen consumption by mitochondrial alternative oxidases (AOXs). AOXs act to prevent overreduction of the mitochondrial electron transport chain. Previously, we reported that AOX1A, the main AOX isoform, is a key component in the developmental response to mitochondrial respiration deficiency. In this work, we suggest the involvement of AOX1A in the response to PGE dysfunction and propose the importance of signaling between plastids and mitochondria. Finally, it was found that seedling architecture reprogramming in response to RIF was independent of canonical organelle retrograde pathways and the ethylene signaling pathway.

Structural basis for the activation and suppression of transposition during evolution of the RAG recombinase.

Jawed vertebrate adaptive immunity relies on the RAG1/RAG2 (RAG) recombinase, a domesticated transposase, for assembly of antigen receptor genes. Using an integration-activated form of RAG1 with methionine at residue 848 and cryo-electron microscopy, we determined structures that capture RAG engaged with transposon ends and U-shaped target DNA prior to integration (the target capture complex) and two forms of the RAG strand transfer complex that differ based on whether target site DNA is annealed or dynamic. Target site DNA base unstacking, flipping, and melting by RAG1 methionine 848 explain how this residue activates transposition, how RAG can stabilize sharp bends in target DNA, and why replacement of residue 848 by arginine during RAG domestication led to suppression of transposition activity. RAG2 extends a jawed vertebrate-specific loop to interact with target site DNA, and functional assays demonstrate that this loop represents another evolutionary adaptation acquired during RAG domestication to inhibit transposition. Our findings identify mechanistic principles of the final step in cut-and-paste transposition and the molecular and structural logic underlying the transformation of RAG from transposase to recombinase.

Self-Adhesive Electronic Skin with Bio-Inspired 3D Architecture for Mechanical Stimuli Monitoring and Human-Machine Interactions.

Recent development of wearable devices is revolutionizing the way of artificial electronic skins (E-skin), physiological health monitoring and human-machine interactions (HMI). However, challenge remains to fit flexible electronic devices to the human skin with conformal deformation and identifiable electrical feedback according to the mechanical stimuli. Herein, an adhesive E-skin is developed that can firmly attach on the human skin for mechanical stimuli perception. The laser-induced adhesive layer serves as the essential component to ensure the conformal attachment of E-skin on curved surface, which ensures the accurate conversion from mechanical deformation to precise electrical readouts. Especially, the 3D architecture facilitates the non-overlapping outputs that bi-directional joint bending and distinguishes strain/pressure. The optimized E-skin with bio-inspired micro-cilia exhibited significantly improved sensing performances with sensitivity of 0.652 kPa-1 in 0-4 kPa and gauge factor of 8.13 for strain (0-15%) with robustness. Furthermore, the adhesive E-skin can distinguish inward/outward joint bending in non-overlapping behaviors, allowing the establishment of ternary system to expand communication capacity for logic outputs such as effective Morse code and intelligent control. It expects that the adhesive E-skin can serve as a functional bridge between human and electrical terminals for applications from daily mechanical monitoring to efficient HMI.

Large Local Internal Stress in an Elastically Bent Molecular Crystal Revealed by Raman Shifts.

The structural dynamics involved in the mechanical flexibility of molecular crystals and the internal stress in such flexible materials remain obscure. Here, the study reports an elastically bending lipidated molecular crystal that shows systematic shifts in characteristic vibrational frequencies across the bent crystal region - revealing the nature of structural changes during bending and the local internal stress distribution. The blueshifts in the bond stretching modes (such as C═O and C-H modes) in the inner arc region and redshifts in the outer arc region of the bent crystals observed via micro-Raman mapping are counterintuitive to the bending models based on intermolecular hydrogen bonds. Correlating these shifts with the trends observed from high-pressure Raman studies on the crystal reveals the local stress difference between the inner arc and outer arc regions of the bent crystal to be ≈2 GPa, more than an order of magnitude higher than the previously proposed value in elastically bending crystals. High local internal stress can have direct ramifications on the properties of molecular piezoelectric energy harvesters, actuators, semiconductors, and flexible optoelectronic materials.

Electroactive Bi-Functional Liquid Crystal Elastomer Actuators.

Liquid crystal elastomers (LCEs) with promising applications in the field of actuators and soft robotics are reported. However, most of them are activated by external heating or light illumination. The examples of electroactive LCEs are still limited; moreover, they are monofunctional with one type of deformation (bending or contraction). Here, the study reports on trilayer electroactive LCE (eLCE) by intimate combination of LCE and ionic electroactive polymer device (i-EAD). This eLCE is bi-functional and can perform either bending or contractile deformations by the control of the low-voltage stimulation. By applying a voltage of ±2 V at 0.1 Hz, the redox behavior and associated ionic motion provide a bending strain difference of 0.80%. Besides, by applying a voltage of ±6 V at 10 Hz, the ionic current-induced Joule heating triggers the muscle-like linear contraction with 20% strain for eLCE without load. With load, eLCE can lift a weight of 270 times of eLCE-actuator weight, while keeping 20% strain and affording 5.38 kJ·m-3 work capacity. This approach of combining two smart polymer technologies (LCE and i-EAD) in a single device is promising for the development of smart materials with multiple degrees of freedom in soft robotics, electronic devices, and sensors.

Gas Release as an Efficient Strategy to Tune Mechanical Properties and Thermoresponsiveness of Dynamic Molecular Crystals.

Dynamic molecular crystals combining multiple and finely tunable functionalities are attracting and an increasing attention due to their potential applications in a broad range of fields as efficient energy transducers and stimuli-responsive materials. In this context, a multicomponent organic salt, piperazinium trifluoroacetate (PZTFA), endowed with an unusual multidimensional responsive landscape is reported. Crystals of the salt undergo smooth plastic deformation under mechanical stress and thermo-induced jumping. Furthermore, via controlled crystal bending and release of trifluoroacetic acid from the lattice, which is anticipated from the design of the material, both the mechanical response and the thermoresponsive behavior are efficiently tuned while partially preserving the crystallinity of the system. In particular, mechanical deformation hampers guest release and hence the macroscopic jumping effect, while trifluoroacetic acid release stiffens the crystals. These complex adaptive responses establish a new crystal engineering strategy to gain further control over dynamic organic crystals.

Mirror-centered representation of a focusing hyperbolic mirror for X-ray beamlines.

Conic sections are commonly used in reflective X-ray optics. Hyperbolic mirrors can focus a converging light source and are frequently paired with elliptical or parabolic mirrors in Wolter type configurations. This paper derives the closed-form expression for a mirror-centered hyperbolic shape, with zero-slope at the origin. Combined with the slope and curvature, such an expression facilitates metrology, manufacturing and mirror-bending calculations. Previous works consider ellipses, parabolas, magnifying hyperbolas or employ lengthy approximations. Here, the exact shape function is given in terms of the mirror incidence angle and the source and image distances.

Low-oxygen-induced root bending is altered by phytoglobin1 through mediation of ethylene response factors (ERFs) and auxin signaling.

MAIN CONCLUSION: phytoglobin1 positively regulates root bending in hypoxic Arabidopsis roots through regulation of ethylene response factors and auxin transport. Hypoxia-induced root bending is known to be mediated by the redundant activity of the group VII ethylene response factors (ERFVII) RAP2.12 and HRE2, causing changes in polar auxin transport (PAT). Here, we show that phytoglobin1 (Pgb1), implicated in hypoxic adaptation through scavenging of nitric oxide (NO), can alter root direction under low oxygen. Hypoxia-induced bending is exaggerated in roots over-expressing Pgb1 and attenuated in those where the gene is suppressed. These effects were attributed to Pgb1 repressing both RAP2.12 and HRE2. Expression, immunological and genetic data place Pgb1 upstream of RAP2.12 and HRE2 in the regulation of root bending in oxygen-limiting environments. The attenuation of slanting in Pgb1-suppressing roots was associated with depletion of auxin activity at the root tip because of depression in PAT, while exaggeration of root bending in Pgb1-over-expressing roots with the retention of auxin activity. Changes in PIN2 distribution patterns, suggestive of redirection of auxin movement during hypoxia, might contribute to the differential root bending responses of the transgenic lines. In the end, Pgb1, by regulating NO levels, controls the expression of 2 ERFVIIs which, in a cascade, modulate PAT and, therefore, root bending.

Auxin response factors fine-tune lignin biosynthesis in response to mechanical bending in bamboo.

Lignin contributes to plant mechanical properties during bending loads. Meanwhile, phytohormone auxin controls various plant biological processes. However, the mechanism of auxin's role in bending-induced lignin biosynthesis was unclear, especially in bamboo, celebrated for its excellent deformation stability. Here, we reported that auxin response factors (ARF) 3 and ARF6 from Moso bamboo (Phyllostachys edulis (Carrière) J. Houz) directly regulate lignin biosynthesis pathway genes, and affect lignin biosynthesis in bamboo. Auxin and lignin exhibited asymmetric distribution patterns, and auxin promoted lignin biosynthesis in response to bending loads in bamboo. Employing transcriptomic and weighted gene co-expression network analysis approach, we discovered that expression patterns of ARF3 and ARF6 strongly correlated with lignin biosynthesis genes. ARF3 and ARF6 directly bind to the promoter regions of 4-coumarate: coenzyme A ligase (4CL3, 4CL7, and 4CL9) or caffeoyl-CoA O-methyltransferase (CCoAOMT2) genes, pivotal to lignin biosynthesis, and activate their expressions. Notably, the efficacy of this binding hinges on auxin levels. Alternation of the expressions of ARF3 and ARF6 substantially altered lignin accumulation in transgenic bamboo. Collectively, our study shed light on bamboo lignification genetics. Auxin signaling could directly modulate lignin biosynthesis genes to impact plant lignin content.