Geometrical manipulation of complex supramolecular tessellations by hierarchical assembly of amphiphilic platinum(II) complexes

Here we report complex supramolecular tessellations achieved by the directed self-assembly of amphiphilic platinum(II) complexes. Despite the twofold symmetry, these geometrically simple molecules exhibit complicated structural hierarchy in a columnar manner. A possible key to such an order increase is the topological transition into circular trimers, which are noncovalently interlocked by metal···metal and π–π interactions, thereby allowing for cofacial stacking in a prismatic assembly. Another key to success is to use the immiscibility of the tailored hydrophobic and hydrophilic sidechains. Their phase separation leads to the formation of columnar crystalline nanostructures homogeneously oriented on the substrate, featuring an unusual geometry analogous to a rhombitrihexagonal Archimedean tiling. Furthermore, symmetry lowering of regular motifs by design results in an orthorhombic lattice obtained by the coassembly of two different platinum(II) amphiphiles. These findings illustrate the potentials of supramolecular engineering in creating complex self-assembled architectures of soft materials.

Tessellation in two dimensions (2D) is a very old topic in geometry on how one or more shapes can be periodically arranged to fill a Euclidean plane without any gaps. Tessellation principles have been extensively applied in decorative art since the early times. In natural sciences, there has been a growing attention on creating ordered structures with increasingly complex architectures inspired by semi-regular Archimedean tilings (ATs) and quasicrystalline textures on account of their intriguing physical properties (1–5) and biological functions (6). Recent advances in this regard have been achieved in various fields of supramolecular science, including the programmable self-assembly of DNA molecules (7), coordination-driven assembly (8–10), supramolecular interfacial engineering (11–13), crystallization of organic polygons (14, 15), colloidal particle superlattices (16), and other soft-matter systems (17–20). Moreover, tessellation in 2D can overcome the topological frustration to generate complex semi- or non-regular patterns by using geometrically simple motifs. As exemplified by the self-templating assembly of spherical soft microparticles (21), a vast array of 2D micropatterns encoding non-regular tilings, such as rectangular, rhomboidal, hexagonal, and herringbone superlattices were obtained by layer-by-layer strategy at a liquid–liquid interface. Tessellation principles have also been extended to the self-assembly of giant molecules in three dimensions (3D). Superlattices with high space-group symmetry (Im3¯m, Pm3¯n, and P4₂/mnm) were reported in dendrimers and dendritic polymers by Percec and coworkers (22–24). Recently, Cheng and coworkers identified the highly ordered Frank–Kasper phases obtained from giant amphiphiles containing molecular nanoparticles (25–28). Despite such advancements made in the field of soft matter, an understanding of how structural ordering in supramolecular materials is influenced by the geometric factors of its constituent molecules has so far remained elusive.In light of these developments and the desire to explore the supramolecular systems, square-planar platinum(II) (Pt^II) polypyridine complexes may serve as an ideal candidate for model studies not only because of their intriguing spectroscopic and luminescence properties (29, 30), but also because of their propensity to form supramolecular polymers or oligomers via noncovalent Pt···Pt and π–π interactions (31–39). Although rod-shaped and lamellar structures are the most commonly observed in the self-assembly of planar Pt^II complexes (34–39), 2D-ordered nanostructures, such as the hexagonally packed columns (31, 40) and honeycomb-like networks (41–43), were recently first demonstrated by us.Herein, we report a serendipitous discovery of a C_2h-symmetric Pt^II amphiphile (Fig. 1A) that can hierarchically self-assemble into a 3D-ordered nanostructure with hexagonal geometry. Interestingly, this structurally anisotropic molecule possibly undergoes topological transition and interlocks to form its circular trimer by noncovalent Pt···Pt and π–π interactions (Fig. 1B). The resultant triangular motif is architecturally stabilized and preorganized for one-dimensional (1D) prismatic assembly (Fig. 1C). Together with the phase separation of the tailored hydrophobic and hydrophilic sidechains, an unusual and unique 3D hexagonal lattice is formed (Fig. 1D), in which the Pt centers adopt a rare rhombitrihexagonal AT-like order. Finally, the nanoarchitecture develops in a hierarchical manner on the substrate due to the homogeneous nucleation (Fig. 1E).Open in a separate windowFig. 1.Hierarchical self-assembly of Pt^II amphiphile into hexagonal ordering. (A) Space-filling (CPK) model of a C_2h-symmetric Pt^II amphiphile (1). All of the hydrogen atoms and counterions are omitted for clarity. (B) CPK representations of possible models of regular triangular, tetragonal, pentagonal, and hexagonal motifs formed with Pt···Pt and π–π stacking. These motifs possess a hydrophilic core (red) with various diameters wrapped by a hydrophobic shell comprising long alkyl chains (gray). (C) CPK representation of a 1D prismatic structure consisting of circular trimers with long-range Pt···Pt and π–π stacking. (D) CPK representation of a 3D columnar lattice constructed by the prismatic assemblies adopting a rare rhombitrihexagonal AT-like order. With the assistance of the phase separation, the hydrophobic domain serves as a discrete column associated with six prismatic neighbors. (E) Schematic representation of the nanoarchitecture with homogeneous orientation.     

Observation of non-Hermitian topology and its bulk–edge correspondence in an active mechanical metamaterial

Topological edge modes are excitations that are localized at the materials’ edges and yet are characterized by a topological invariant defined in the bulk. Such bulk–edge correspondence has enabled the creation of robust electronic, electromagnetic, and mechanical transport properties across a wide range of systems, from cold atoms to metamaterials, active matter, and geophysical flows. Recently, the advent of non-Hermitian topological systems—wherein energy is not conserved—has sparked considerable theoretical advances. In particular, novel topological phases that can only exist in non-Hermitian systems have been introduced. However, whether such phases can be experimentally observed, and what their properties are, have remained open questions. Here, we identify and observe a form of bulk–edge correspondence for a particular non-Hermitian topological phase. We find that a change in the bulk non-Hermitian topological invariant leads to a change of topological edge-mode localization together with peculiar purely non-Hermitian properties. Using a quantum-to-classical analogy, we create a mechanical metamaterial with nonreciprocal interactions, in which we observe experimentally the predicted bulk–edge correspondence, demonstrating its robustness. Our results open avenues for the field of non-Hermitian topology and for manipulating waves in unprecedented fashions.

The inclusion of non-Hermitian features in topological insulators has recently seen an explosion of activity. Exciting developments include tunable wave guides that are robust to disorder (1–3), structure-free systems (4, 5), and topological lasers and pumping (6–10). In these systems, active components are introduced to typically 1) break time-reversal symmetry to create topological insulators with unidirectional edge modes (1–5) and 2) pump topologically protected edge modes, thus harnessing Hermitian topology in non-Hermitian settings (6–9, 11). In parallel, extensive theoretical efforts have generalized the concept of a topological insulator to truly non-Hermitian phases that cannot be realized in Hermitian materials (12–14). However, such non-Hermitian topology and its bulk–edge correspondence remain a matter of intense debate. On the one hand, it has been argued that the usual bulk–edge correspondence breaks down in non-Hermitian settings, while on the other hand, new topological invariants specific to non-Hermitian systems have been proposed to capture particular properties of their edge modes (15–20).Here, focusing on a non-Hermitian version of the Su–Schrieffer–Heeger (SSH) model (15–17, 21) with an odd number of sites (Fig. 1A), we find that a change in the bulk non-Hermitian topological invariant is accompanied by a localization change in the zero-energy edge modes. This finding suggests the existence of a bulk–edge correspondence for this type of truly non-Hermitian topology. We further construct a mechanical analogue of the non-Hermitian quantum model (Fig. 1B) and create a mechanical metamaterial (Fig. 1C) in which we observe the predicted correspondence between the non-Hermitian topological invariant and the topological edge mode. In particular, we report that the edge mode in the non-Hermitian topological phase has a peculiar nature, as it is localized on the rigid rather than the floppy side of the mechanical metamaterial.Open in a separate windowFig. 1.Quantum-to-classical mapping of a chain with non-Hermitian topology. (A) An SSH chain with two sublattices, A (in red) and B (in blue), augmented with nonreciprocal variations in the hopping amplitudes (indicated by ±ε). (B) The nonreciprocal classical analog of the augmented SSH chain, in which the classical masses (in red) correspond to the A sites in the quantum model, while the nonreciprocal springs (in blue) are analogous to the B sites. (C) Picture of the mechanical metamaterial realizing the nonreciprocal classical analogue of the augmented SSH model.     

On and off controls within dynein–dynactin on native cargoes

The dynein–dynactin nanomachine transports cargoes along microtubules in cells. Why dynactin interacts separately with the dynein motor and also with microtubules is hotly debated. Here we disrupted these interactions in a targeted manner on phagosomes extracted from cells, followed by optical trapping to interrogate native dynein–dynactin teams on single phagosomes. Perturbing the dynactin–dynein interaction reduced dynein’s on rate to microtubules. In contrast, perturbing the dynactin–microtubule interaction increased dynein’s off rate markedly when dynein was generating force against the optical trap. The dynactin–microtubule link is therefore required for persistence against load, a finding of importance because disease-relevant mutations in dynein–dynactin are known to interfere with “high-load” functions of dynein in cells. Our findings call attention to a less studied property of dynein–dynactin, namely, its detachment against load, in understanding dynein dysfunction.

Transport of organelles inside cells is driven by motors of the kinesin and dynein families that generate force, respectively, toward the plus and minus ends of microtubules (MTs). Cytoplasmic dynein, aided by many regulators such as dynactin (Fig. 1A), executes bewilderingly diverse cellular functions (1). Dynactin’s largest subunit P150 has a coiled-coil (CC) region that contains a cytoskeleton-associated protein glycine-rich (CAPGly) domain along with a stretch of basic residues (2). Dynactin binds dynein through its CC1 domain, and dynactin also binds MTs through its CAPGly and basic domains (Fig. 1A). Additional interactions between dynein heavy chain and dynactin’s Arp domain may stabilize the complex (3, 4). How all these linkages help dynein–dynactin to function is hotly debated (5, 6). Of particular interest is the recruitment of dynein–dynactin to cargoes by different adaptor proteins (1), as also revealed by the cryogenic electron microscopy (cryo-EM) structure of dynein–dynactin–adaptor complexes wherein two dimeric dynein motors were found to be recruited as a pair (3, 4). Dynein undergoes significant conformational changes in this process, allowing both motor domains to align along the MT for effective force generation. Notably, recruitment of dynein in pairs to phagosomes was suggested by us on the basis of force measurements inside cells (7).Open in a separate windowFig. 1.Effects of DIC-WT on dynein-membrane and dynein–MT binding. (A) (Left) Schematic of a single phagosome driven by a team of dyneins and a kinesin held in an optical trap over a MT. (Right) Magnified schematic of one dynein–dynactin complex. Treatments to target the DIC–CC1 interaction (with recombinant DIC protein) and CAPGly–MT interaction (with anti-CAPGly antibody) are shown. DIC, dynein intermediate chain; DHC, dynein heavy chain; ARP, actin-related protein. (B) Western blot showing levels of dynein (DIC) and dynactin (P150) retained on organelle membranes after treatment with mDIC-WT or mDIC-Mut. Membranes were prepared from RAW mouse macrophage cells. Rab7 is a marker for late endosomes. Actin is a loading control. Lower panel shows quantification of dynein band intensity across three experiments. The mDIC-Mut band intensity was always taken equal to 1 and mDIC-WT intensity calculated relative to mDIC-Mut. No significant difference is seen between mDIC-WT and mDIC-Mut treatments. Error bars, SEM. P value was calculated using Student’s t test. (C) Goat brain cytosol was treated with mDIC-WT or mDIC-Mut, followed by addition of exogenously polymerized MTs. Western blot shows levels of dynein (DIC) and dynactin (P150) that copelleted with MTs. Tubulin from the resuspended MT pellet is used for normalization. Lower panel shows quantification across three experiments. The mDIC-Mut band intensity was always taken equal to 1 and mDIC-WT intensity calculated relative to mDIC-Mut. Dynein and dynactin binding to MTs is reduced upon addition of mDIC-WT. Error bars, SEM. P values were calculated using Student’s t test.Two kinds of studies, classified here as in vitro and in vivo, have investigated dynein–dynactin function. In vitro, dynactin was suggested to work as a brake for dynein (8); however, others found dynactin to enhance processive motion of dynein-driven beads (2, 9, 10). Purified dynein and dynactin, when complexed with bicaudal-D (Bic-D) activated dynein for motion (3, 11–14). An elegant demonstration of such activation came from McKenney et al. (12, 15) and Schlager et al. (14). MT binding by dynactin was suggested to initiate processive transport along tyrosinated MTs in vitro by reducing detachment of the dynein–dynactin complex from MTs (12, 15). In vivo studies have relied on overexpression or genetic perturbation of dynactin subunits inside cells. Studies targeting the dynactin–MT binding in neurons suggested that dynactin is not needed once retrograde transport from neurite tips has been initiated (16, 17). Perturbing dynactin–MT binding had no effect on processivity and step size of dynein, but affected MT organization in cells (18). In vitro, however, a CAPGly antibody reduced run length of dynein–dynactin-driven motion (9, 19) and loss of the CAPGly domain increased dynein detachment from MTs (8). The in vitro assays described above are bereft of many (known and unknown) protein regulators of dynein. Further, native-like function requires motors to be assembled on a lipid membrane on the cargo (20), a component missing in most in vitro assays. On the other hand, in vivo studies often cannot extract mechanistic details of function at the single-cargo level and could also suffer from off-target effects arising from genetic perturbations that are introduced in the cells.To address these controversies, we fed micrometer-sized latex beads into Dictyostelium cells and allowed ingested beads to mature into phagosomes, wherein dynein and kinesin assembled in situ on the phagosome membrane. Phagosomes were then extracted from cells and motion and force generation of single phagosomes against an optical trap were interrogated ex vivo on polarity-labeled MTs (Fig. 1A). Most parameters of phagosome motion inside cells (7) were replicated in this ex vivo assay (21). Recombinant proteins and antibodies were then used to induce targeted perturbation of specific interactions within the dynein–dynactin machinery on phagosomes ex vivo (Fig. 1A). The consequence of these targeted perturbations was assayed by optical trapping and biochemical studies. Lastly, we reconstituted endogenous dynein–dynactin complexes on a lipid membrane that was assembled artificially on a bead (called supported lipid bilayer [SLB]). SLBs exhibit vigorous dynein–dynactin-driven motion, and targeted perturbation of dynein–dynactin on the SLBs replicates the effects seen on phagosomes. These SLB assays establish that the perturbations and their effects are indeed specific to the dynein–dynactin complex.Perturbing the interaction between dynein intermediate chain (DIC) and the CC1 domain of dynactin reduces the binding rate (K_ON) of dynein–dynactin to MTs, but has little effect on the detachment rate (K_OFF) of dyneins from the MT. In contrast, perturbing the dynactin–MT interaction increases K_OFF most appreciably when dynein attempts to move against opposing force (i.e., load) exerted by an optical trap. Therefore, the DIC–CC1 interaction maintains dynein in an initial (unloaded) conformation that binds rapidly to MTs (high K_ON) to initiate motion. Once motion is initiated, the dynactin–MT interaction ensures tenacious binding (low K_OFF) of dynein–dynactin to MTs against opposing force, allowing dynein–dynactin to compete against other motors and/or obstacles. We suggest a mechanism wherein a load-dependent communication operates across the dynein–dynactin scaffold and is necessary for the nanomachine to generate persistent force against opposition.     

Stable metal anodes enabled by a labile organic molecule bonded to a reduced graphene oxide aerogel

Metallic anodes (lithium, sodium, and zinc) are attractive for rechargeable battery technologies but are plagued by an unfavorable metal–electrolyte interface that leads to nonuniform metal deposition and an unstable solid–electrolyte interphase (SEI). Here we report the use of electrochemically labile molecules to regulate the electrochemical interface and guide even lithium deposition and a stable SEI. The molecule, benzenesulfonyl fluoride, was bonded to the surface of a reduced graphene oxide aerogel. During metal deposition, this labile molecule not only generates a metal-coordinating benzenesulfonate anion that guides homogeneous metal deposition but also contributes lithium fluoride to the SEI to improve Li surface passivation. Consequently, high-efficiency lithium deposition with a low nucleation overpotential was achieved at a high current density of 6.0 mA cm⁻². A Li|LiCoO₂ cell had a capacity retention of 85.3% after 400 cycles, and the cell also tolerated low-temperature (−10 °C) operation without additional capacity fading. This strategy was applied to sodium and zinc anodes as well.

Rechargeable batteries based on metal anodes including lithium (Li), sodium (Na), and zinc (Zn) show great promise in achieving high energy density (1–3). Unfortunately, the electrochemical interface of the metal anodes is not favorable for metal deposition. Metal nucleation is inhomogeneous at the surface, leading to the growth of metal dendrites (4–7) and the formation of an unstable solid–electrolyte interphase (SEI) that is incapable of protecting metals from the side reactions with the electrolyte (8–12).Substantial efforts have been devoted to stabilizing the interface of metal anodes, especially for Li metal. These include the design of artificial protective layers (13–17), alternative electrolytes (18–24), and sacrificial additives (25–30) to stabilize the metal–electrolyte interface, the development of mechanically robust coatings (31–34) to block Li dendrite growth, and the use of structured scaffolds to host dendrite-free Li deposition by reducing local current densities (35–43). However, the performance of metal anodes remains poor under high-current or low-temperature conditions. This is because the inhomogeneous Li nucleation and unstable SEI problems have not been well addressed, and these problems at the interface are even exacerbated under critical operating conditions, especially high-current densities and low temperatures (5, 6, 44).Toward this end, we report a simple molecular approach for regulating the electrochemical interface of metal anodes, which enables even Li deposition and stable SEI formation in a conventional electrolyte. This was realized by bonding a labile organic molecule, benzenesulfonyl fluoride (BSF), to a reduced graphene oxide (rGO) aerogel surface as the Li anode host (Fig. 1A). During Li deposition, BSF molecules electrochemically decompose at the interface and generate benzenesulfonate anions bonded to the rGO aerogel (Fig. 1B). The conjugated anions have a strong binding affinity for Li, serving as lithiophilic sites on the rGO surface to synergistically induce homogeneous Li nucleation of Li on the rGO surface. At the same time, BSF molecules contribute LiF to the SEI layer, which facilitates Li surface passivation (Fig. 1C). As a result, high-efficiency (99.2%) Li deposition was achieved at a Li deposition amount of 6.0 mAh cm⁻² and a current density of 6.0 mA cm⁻²; the barrier to Li nucleation was markedly reduced, as evidenced by the low nucleation overpotentials at high-current density (6.0 mA cm⁻²) or at a low temperature (−10 °C). A 400-cycle life with a capacity retention of 83.6% was achieved for a Li|LiCoO₂ (LCO) cell in a conventional carbonate electrolyte. Moreover, with the organic molecule-tuned interface, the Li|LCO cell can be stably cycled at a low operating temperature (−10 °C). This approach was applied to Na and Zn metal anodes as well.Open in a separate windowFig. 1.Illustration of a stable interface for Li deposition using a labile organic molecule, benzenesulfonyl fluoride (BSF). (A) Covalently bonded BSF on the rGO aerogel surface. (B) In situ generation of a lithiophilic conjugated anion (benzenesulfonate) and LiF on the surface during Li deposition. (C) Li nucleation preferentially occurs at the conjugated anion sites owing to the strong Li binding affinity, which leads to uniform Li deposition. In addition, the LiF that is formed is in the SEI layer and passivates the Li surface.     

Trapping a cross-linked lysine–tryptophan radical in the catalytic cycle of the radical SAM enzyme SuiB

The radical S-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon–carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys–Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]²⁺ state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys–Trp radical (Lys–Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys–Trp• intermediate by the redox-tuned [4Fe-4S]²⁺ AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon–carbon bond-forming reactions.

The radical S-adenosylmethionine (rSAM) enzyme superfamily is the largest known in nature, with over 570,000 annotated and predominantly uncharacterized members spanning all domains of life (1–4). The uniting feature of rSAM enzymes is a [4Fe-4S] cluster, usually bound by a CX₃CX₂C motif that catalyzes reductive cleavage of SAM to form L-Met and a strongly oxidizing 5′-deoxyadenosyl radical (5′-dA•) (5–7). Recent studies on a suite of rSAM enzymes have revealed the presence of a previously unknown organometallic intermediate in this process, termed Ω, in which the 5′-C of 5′-dA• is bound to the unique iron of the [4Fe-4S] cluster (Fig. 1A) (8, 9). Homolysis of the Fe–C bond ultimately liberates 5′-dA•, which abstracts a hydrogen atom from substrate to initiate a profoundly diverse set of chemical reactions in both primary and secondary metabolism, including DNA, cofactor, vitamin, and antibiotic biosynthesis (5, 10–13).Open in a separate windowFig. 1.(A) Accepted scheme for radical initiation in rSAM enzymes. (B) X-ray crystal structure of SuiB (PDB ID: 5V1T). The RS domain, SPASM domain, and RiPP recognition element are rendered blue, green, and pink, respectively. [4Fe-4S] clusters are shown as spheres with the distances separating them indicated. (C) Lys–Trp cross-link formation (20) catalyzed by SuiB. The carbon–carbon bond installed by SuiB is shown in red. (D and E) Previously proposed EAS (D) and rEAS (E) mechanisms for SuiB-catalyzed Lys–Trp cross-link formation.Of the 570,000 rSAM enzyme superfamily members, over a quarter (∼165,000 genes from the Enzyme Function Initiative-Enzyme Similarity Tool) possess C-terminal extensions, called SPASM and twitch domains, which bind auxiliary Fe-S clusters (4, 14–19). The SPASM domain typically binds two auxiliary Fe-S clusters and is named after the rSAM enzymes involved in the synthesis of subtilosin, pyrroloquinoline quinone, anaerobic sulfatase, and mycofactocin. The twitch domain is a truncated SPASM domain and only binds one auxiliary cluster (15). Despite the wide prevalence of these domains and the characterization of several different SPASM/twitch rSAM enzymes by spectroscopic and structural studies, direct evidence for their catalytic function(s) has remained elusive.We previously performed functional and structural characterization on the SPASM rSAM enzyme SuiB (Fig. 1B), which is involved in the biosynthesis of a ribosomal peptide natural product in human and mammalian microbiome streptococci (14, 20–22). SuiB introduces an unusual carbon–carbon bond onto its substrate peptide, SuiA, between the sidechains of Lys2 and Trp6 (Fig. 1C). The mechanism for this transformation is of broader relevance, as a number of enzymes, such as RrrB, PqqE, and MqnC (2, 23, 24), are known to join unactivated aliphatic and aromatic carbons to generate sp³-sp² cross-links. A general mechanistic paradigm for this class of transformations is not yet available. For SuiB, two pathways have been proposed (20), one through a typical electrophilic aromatic substitution (EAS) mechanism, which is involved in other enzyme-catalyzed indole modifications, such as indole prenylation or flavin adenine dinucleotide (FAD)-enzyme-dependent indole chlorination (25–27). In this pathway, the 5′-dA• generates an alkyl radical, which upon a second one-electron oxidation, creates an α,β-unsaturated amide electrophile with which the indole sidechain reacts via Michael addition (Fig. 1D). Lanthionine cross-links observed in diverse lanthipeptides are built by this general scheme, though via heterolytic chemistry, with Cys acting as the nucleophile (28, 29). Alternatively, a radical electrophilic aromatic substitution (rEAS) reaction has been proposed, wherein the alkyl radical, formed by 5′-dA•, would react with the indole sidechain to generate a radical σ complex, a cross-linked Lys–Trp radical (Lys–Trp•), which upon oxidation and rearomatization would yield product (Fig. 1E). In both mechanisms, AuxI is proposed as an oxidant. Although this role for an rSAM auxiliary cluster has been previously suggested (30, 31), it has yet to be directly demonstrated experimentally. Mechanistic studies have favored the rEAS pathway (20); however, intermediates in the reaction of SuiB and enzymes that catalyze similar reactions have not yet been detected (15).In the current work, we sought to differentiate between the proposed mechanisms by trapping intermediates in the catalytic cycle of SuiB and characterizing them using electron paramagnetic resonance (EPR) spectroscopy. We report observation of three transient reaction intermediates, most importantly the sought-after Lys–Trp•, which is fundamentally different from previously characterized Trp radicals, as it is cross-linked and carries an indole tetrahedral center. We also provide evidence for AuxI as the oxidant of the Lys–Trp• intermediate as well as insights into redox potential changes of Fe-S clusters in SuiB that accompany SuiA binding. Together, our findings support the rEAS pathway for formation of the sp³-sp² cross-link and carry important implications for other enzymes that catalyze related transformations.     

A rate threshold mechanism regulates MAPK stress signaling and survival

Cells are exposed to changes in extracellular stimulus concentration that vary as a function of rate. However, how cells integrate information conveyed from stimulation rate along with concentration remains poorly understood. Here, we examined how varying the rate of stress application alters budding yeast mitogen-activated protein kinase (MAPK) signaling and cell behavior at the single-cell level. We show that signaling depends on a rate threshold that operates in conjunction with stimulus concentration to determine the timing of MAPK signaling during rate-varying stimulus treatments. We also discovered that the stimulation rate threshold and stimulation rate-dependent cell survival are sensitive to changes in the expression levels of the Ptp2 phosphatase, but not of another phosphatase that similarly regulates osmostress signaling during switch-like treatments. Our results demonstrate that stimulation rate is a regulated determinant of cell behavior and provide a paradigm to guide the dissection of major stimulation rate dependent mechanisms in other systems.

All cells employ signal transduction pathways to respond to physiologically relevant changes in extracellular stressors, nutrient levels, hormones, morphogens, and other stimuli that vary as functions of both concentration and rate in healthy and diseased states (1–7). Switch-like “instantaneous” changes in the concentrations of stimuli in the extracellular environment have been widely used to show that the strength of signaling and overall cellular response are dependent on the stimulus concentration, which in many cases needs to exceed a certain threshold (8, 9). Previous studies have shown that the rate of stimulation can also influence signaling output in a variety of pathways (10–17) and that stimulation profiles of varying rates can be used to probe underlying signaling pathway circuitry (4, 18, 19). However, it is still not clear how cells integrate information conveyed by changes in both the stimulation rate and concentration in determining signaling output. It is also not clear if cells require stimulation gradients to exceed a certain rate in order to commence signaling.Recent investigations have demonstrated that stimulation rate can be a determining factor in signal transduction. In contrast to switch-like perturbations, which trigger a broad set of stress-response pathways, slow stimulation rates activate a specific response to the stress applied in Bacillus subtilis cells (10). Meanwhile, shallow morphogen gradient stimulation fails to activate developmental pathways in mouse myoblast cells in culture, even when concentrations sufficient for activation during pulsed treatment are delivered (12). These observations raise the possibility that stimulation profiles must exceed a set minimum rate or rate threshold to achieve signaling activation. Although such rate thresholds would help cells decide if and how to respond to dynamic changes in stimulus concentration, the possibility of signaling regulation by a rate threshold has never been directly investigated in any system. Further, no study has experimentally examined how stimulation rate requirements impact cell phenotype or how cells molecularly regulate the stimulation rate required for signaling activation. As such, the biological significance of any existing rate threshold regulation of signaling remains unknown.The budding yeast Saccharomyces cerevisiae high osmolarity glycerol (HOG) pathway provides an ideal model system for addressing these issues (Fig. 1A). The evolutionarily conserved mitogen-activated protein kinase (MAPK) Hog1 serves as the central signaling mediator of this pathway (20–22). It is well established that instantaneous increases in osmotic stress concentration induce Hog1 phosphorylation, activation, and translocation to the nucleus (18, 21, 23–30). Activated Hog1 governs the majority of the cellular osmoadaptation response that enables cells to survive (23, 31, 32). Multiple apparently redundant MAPK phosphatases dephosphorylate and inactivate Hog1, which, along with the termination of upstream signaling after adaptation, results in its return to the cytosol (Fig. 1A) (23, 25, 26, 33–39). Because of this behavior, time-lapse analysis of Hog1 nuclear enrichment in single cells has proven an excellent and sensitive way to monitor signaling responses to dynamic stimulation patterns in real time (18, 27–30, 40, 41). Further, such assays have been readily combined with traditional growth and molecular genetic approaches to link observed signaling responses with cell behavior and signaling pathway architecture (27–29).Open in a separate windowFig. 1.Hog1 signaling and cell survival are sensitive to the rate of preconditioning osmotic stress application. (A) Schematic of the budding yeast HOG response. (B) Preconditioning protection assay workflow indicating the first stress treatments to a final concentration of 0.4 M NaCl (Left), high-stress exposure (Middle), and colony formation readout (Right). (C) High-stress survival as a function of each first treatment relative to the untreated first stress condition. Bars and errors are means and SD from three biological replicates. *Statistically significant by Kolmogorov–Smirnov test (P < 0.05). NS = not significant. (D) Treatment concentration over time. (E) Treatment rate over time for quadratic and pulse treatment. The rate for the pulse is briefly infinite (blue vertical line) before it drops to 0. (F) Hog1 nuclear localization during the treatments depicted in D and E. (Inset) Localization pattern in the quadratic-treated sample. Lines represent means and shaded error represents the SD from three to four biological replicates.Here, we use systematically designed osmotic stress treatments imposed at varying rates of increase to show that a rate threshold condition regulates yeast high-stress survival and Hog1 MAPK signaling. We demonstrate that only stimulus profiles that satisfy both this rate threshold condition and a concentration threshold condition result in robust signaling. We go on to show that the protein tyrosine phosphatase Ptp2, but not the related Ptp3 phosphatase, serves as a major rate threshold regulator. By expressing PTP2 under the control of a series of different enhancer–promoter DNA constructs, we demonstrate that changes in the level of Ptp2 expression can alter the stimulation rate required for signaling induction and survival. These findings establish rate thresholds as a critical and regulated component of signaling biology akin to concentration thresholds.     

Paleocene latitude of the Kohistan–Ladakh arc indicates multistage India–Eurasia collision

We report paleomagnetic data showing that an intraoceanic Trans-Tethyan subduction zone existed south of the Eurasian continent and north of the Indian subcontinent until at least Paleocene time. This system was active between 66 and 62 Ma at a paleolatitude of 8.1 ± 5.6 °N, placing it 600–2,300 km south of the contemporaneous Eurasian margin. The first ophiolite obductions onto the northern Indian margin also occurred at this time, demonstrating that collision was a multistage process involving at least two subduction systems. Collisional events began with collision of India and the Trans-Tethyan subduction zone in Late Cretaceous to Early Paleocene time, followed by the collision of India (plus Trans-Tethyan ophiolites) with Eurasia in mid-Eocene time. These data constrain the total postcollisional convergence across the India–Eurasia convergent zone to 1,350–2,150 km and limit the north–south extent of northwestern Greater India to <900 km. These results have broad implications for how collisional processes may affect plate reconfigurations, global climate, and biodiversity.

Classically, the India–Eurasia collision has been considered to be a single-stage event that occurred at 50–55 million years ago (Ma) (1, 2). However, plate reconstructions show thousands of kilometers of separation between India and Eurasia at the inferred time of collision (3, 4). Accordingly, the northern extent of Greater India was thought to have protruded up to 2,000 km relative to present-day India (5, 6) (Fig. 1). Others have suggested that the India–Eurasia collision was a multistage process that involved an east–west trending Trans-Tethyan subduction zone (TTSZ) situated south of the Eurasian margin (7–9) (Fig. 1). Jagoutz et al. (9) concluded that collision between India and the TTSZ occurred at 50–55 Ma, and the final continental collision occurred between the TTSZ and Eurasia at 40 Ma (9, 10). This model reconciles the amount of convergence between India and Eurasia with the observed shortening across the India–Eurasia collision system with the addition of the Kshiroda oceanic plate. Additionally, the presence of two subduction systems can explain the rapid India–Eurasia convergence rates (up to 16 mm a⁻¹) that existed between 135 and 50 Ma (9), as well as variations in global climate in the Cenozoic (11).Open in a separate windowFig. 1.The first panel is an overview map of tectonic structure of the Karakoram–Himalaya–Tibet orogenic system. Blue represents India, red represents Eurasia, and the Kohistan–Ladakh arc (KLA) is shown in gray. The different shades of blue highlight the deformed margin of the Indian plate that has been uplifted to form the Himalayan belt, and the zones of darker red within the Eurasian plate highlight the Eurasian continental arc batholith. Thick black lines denote the suture zones which separate Indian and Eurasian terranes. The tectonic summary panels illustrate the two conflicting collision models and their differing predictions of the location of the Kohistan–Ladakh arc. India is shown in blue, Eurasia is shown in red, and the other nearby continents are shown in gray. Active plate boundaries are shown with black lines, and recently extinct boundaries are shown with gray lines. Subduction zones are shown with triangular tick marks.While the existence of the TTSZ in the Cretaceous is not disputed, the two conflicting collision models make distinct predictions about its paleolatitude in Late Cretaceous to Paleocene time; these can be tested using paleomagnetism. In the single-stage collision model, the TTSZ amalgamated with the Eurasian margin prior to ∼80 Ma (12) at a latitude of ≥20 °N (13, 14). In contrast, in the multistage model, the TTSZ remained near the equator at ≤10 °N, significantly south of Eurasia, until collision with India (9) (Fig. 1).No undisputed paleomagnetic constraints on the location of the TTSZ are available in the central Himalaya (15–17). Westerweel et al. (18) showed that the Burma Terrane, in the eastern Himalaya, was part of the TTSZ and was located near the equator at ∼95 Ma, but they do not constrain the location of the TTSZ in the time period between 50 and 80 Ma, which is required to test the two collision hypotheses. In the western Himalaya, India and Eurasia are separated by the Bela, Khost, and Muslimbagh ophiolites and the 60,000 km² intraoceanic Kohistan Ladakh arc (19, 20) (Fig. 1). These were obducted onto India in the Late Cretaceous to Early Paleocene (19), prior to the closure of the Eocene to Oligocene Katawaz sedimentary basin (20) (Fig. 1). The Kohistan–Ladakh arc contacts the Eurasian Karakoram terrane in the north along the Shyok suture and the Indian plate in the south along the Indus suture (21) (Fig. 1). Previous paleomagnetic studies suggest that the Kohistan–Ladakh arc formed as part of the TTSZ near the equator in the early Cretaceous but provide no information on its location after 80 Ma (22–25). While pioneering, these studies lack robust age constraints, do not appropriately average paleosecular variation of the geodynamo, and do not demonstrate that the measured magnetizations have not been reset during a subsequent metamorphic episode.     

From the Cover: Neurophysiological coordination of duet singing

Coordination of behavior for cooperative performances often relies on linkages mediated by sensory cues exchanged between participants. How neurophysiological responses to sensory information affect motor programs to coordinate behavior between individuals is not known. We investigated how plain-tailed wrens (Pheugopedius euophrys) use acoustic feedback to coordinate extraordinary duet performances in which females and males rapidly take turns singing. We made simultaneous neurophysiological recordings in a song control area “HVC” in pairs of singing wrens at a field site in Ecuador. HVC is a premotor area that integrates auditory feedback and is necessary for song production. We found that spiking activity of HVC neurons in each sex increased for production of its own syllables. In contrast, hearing sensory feedback produced by the bird’s partner decreased HVC activity during duet singing, potentially coordinating HVC premotor activity in each bird through inhibition. When birds sang alone, HVC neurons in females but not males were inhibited by hearing the partner bird. When birds were anesthetized with urethane, which antagonizes GABAergic (γ-aminobutyric acid) transmission, HVC neurons were excited rather than inhibited, suggesting a role for GABA in the coordination of duet singing. These data suggest that HVC integrates information across partners during duets and that rapid turn taking may be mediated, in part, by inhibition.

Animals routinely rely on sensory feedback for the control of their own behavior. In cooperative performances, such sensory feedback can include cues produced by other participants (1–8). For example, in interactive vocal communication, including human speech, individuals take turns vocalizing. This “turn taking” is a consequence of each participant responding to auditory cues from a partner (4–6, 9, 10). The role of such “heterogenous” (other-generated) feedback in the control of vocal turn taking and other cooperative performances is largely unknown.Plain-tailed wrens (Pheugopedius euophrys) are neotropical songbirds that cooperate to produce extraordinary duet performances but also sing by themselves (Fig. 1A) (4, 10, 11). Singing in plain-tailed wrens is performed by both females and males and used for territorial defense and other functions, including mate guarding and attraction (1, 11–16). During duets, female and male plain-tailed wrens take turns, alternating syllables at a rate of between 2 and 5 Hz (Fig. 1A) (4, 11).Open in a separate windowFig. 1.Neural control of solo and duet singing in plain-tailed wrens. (A) Spectrogram of a singing bout that included male solo syllables (blue line, top) followed by a duet. Solo syllables for both sexes (only male solo syllables are shown here) are sung at lower amplitudes than syllables produced in duets. Note that the smeared appearance of wren syllables in spectrograms reflects the acoustic structure of plain-tailed wren singing. (B and C) Each bird has a motor system that is used to produce song and sensory systems that mediate feedback. (B) During solo singing, the bird hears its own song, which is known as autogenous feedback (orange). (C) During duet singing, each bird hears both its own singing and the singing of its partner, known as heterogenous feedback (green). The key difference between solo and duet singing is heterogenous feedback that couples the neural systems of the two birds. This coupling results in changes in syllable amplitude and timing in both birds.There is a categorical difference between solo and duet singing. In solo singing, the singing bird receives only autogenous (hearing its own vocalization) feedback (Fig. 1B). The partner may hear the solo song if it is nearby, a heterogenous (other-generated) cue. In duet singing, birds receive both heterogenous and autogenous feedback as they alternate syllable production (Fig. 1C). Participants use heterogenous feedback during duet singing for precise timing of syllable production (4, 11). For example, when a male temporarily stops participating in a duet, the duration of intersyllable intervals between female syllables increases (4), showing an effect of heterogenous feedback on the timing of syllable production.How does the brain of each wren integrate heterogenous acoustic cues to coordinate the precise timing of syllable production between individuals during duet performances? To address this question, we examined neurophysiological activity in HVC, a nucleus in the nidopallium [an analogue of mammalian cortex (17, 18)]. HVC is necessary for song learning, production, and timing in species of songbirds that do not perform duets (19–24). Neurons in HVC are active during singing and respond to playback of the bird’s own learned song (25–27). In addition, recent work has shown that HVC is also involved in vocal turn taking (19).To examine the role of heterogenous feedback in the control of duet performances, we compared neurophysiological activity in HVC when female or male wrens sang solo syllables with syllables sung during duets. Neurophysiological recordings were made in awake and anesthetized pairs of wrens at the Yanayacu Biological Station and Center for Creative Studies on the slopes of the Antisana volcano in Ecuador. We found that heterogenous cues inhibited HVC activity during duet performances in both females and males, but inhibition was only observed in females during solo singing.     

Heading perception depends on time-varying evolution of optic flow

There is considerable support for the hypothesis that perception of heading in the presence of rotation is mediated by instantaneous optic flow. This hypothesis, however, has never been tested. We introduce a method, termed “nonvarying phase motion,” for generating a stimulus that conveys a single instantaneous optic flow field, even though the stimulus is presented for an extended period of time. In this experiment, observers viewed stimulus videos and performed a forced-choice heading discrimination task. For nonvarying phase motion, observers made large errors in heading judgments. This suggests that instantaneous optic flow is insufficient for heading perception in the presence of rotation. These errors were mostly eliminated when the velocity of phase motion was varied over time to convey the evolving sequence of optic flow fields corresponding to a particular heading. This demonstrates that heading perception in the presence of rotation relies on the time-varying evolution of optic flow. We hypothesize that the visual system accurately computes heading, despite rotation, based on optic acceleration, the temporal derivative of optic flow.

James Gibson first remarked that the instantaneous motion of points on the retina (Fig. 1A) can be formally described as a two-dimensional (2D) field of velocity vectors called the “optic flow field” (or “optic flow”) (1). Such optic flow, caused by an observer’s movement relative to the environment, conveys information about self-motion and the structure of the visual scene (1–15). When an observer translates in a given direction along a straight path, the optic flow field radiates from a point in the image with zero velocity, or singularity, called the focus of expansion (Fig. 1B). It is well known that under such conditions, one can accurately estimate one’s “heading” (i.e., instantaneous direction of translation in retinocentric coordinates) by simply locating the focus of expansion (SI Appendix). However, if there is angular rotation in addition to translation (by moving along a curved path or by a head or eye movement), the singularity in the optic flow field will be displaced such that it no longer corresponds to the true heading (Fig. 1 C and D). In this case, if one estimates heading by locating the singularity, the estimate will be biased away from the true heading. This is known as the rotation problem (14).Open in a separate windowFig. 1.Projective geometry, the rotation problem, time-varying optic flow, and the optic acceleration hypothesis. (A) Viewer-centered coordinate frame and perspective projection. Because of motion between the viewpoint and the scene, a 3D surface point traverses a path in 3D space. Under perspective projection, the 3D path of this point projects onto a 2D path in the image plane (retina), the temporal derivative of which is called image velocity. The 2D velocities associated with all visible points define a dense 2D vector field called the optic flow field. (B–D) Illustration of the rotation problem. (B) Optic flow for pure translation (1.5-m/s translation speed, 0° heading, i.e., heading in the direction of gaze). Optic flow singularity (red circle) corresponds to heading (purple circle). (C) Pure rotation, for illustrative purposes only and not corresponding to any experimental condition (2°/s rightward rotation). (D) Translation + rotation (1.5 m/s translation speed, 0° heading, 2°/s rightward rotation). Optic flow singularity (red circle) is displaced away from heading (purple circle). (E) Three frames from a video depicting movement along a circular path with the line-of-sight initially perpendicular to a single fronto-parallel plane composed of black dots. (F) Time-varying evolution of optic flow. The first optic flow field reflects image motion between the first and second frames of the video. The second optic flow field reflects image motion between the second and third frames of the video. For this special case (circular path), the optic flow field evolves (and the optic flow singularity drifts) only due to the changing depth of the environment relative to the viewpoint. (G) Illustration of the optic acceleration hypothesis. Optic acceleration is the derivative of optic flow over time (here, approximated as the difference between the second and first optic flow fields). The singularity of the optic acceleration field corresponds to the heading direction. Acceleration vectors autoscaled for visibility.Computer vision researchers and vision scientists have developed a variety of algorithms that accurately and precisely extract observer translation and rotation from optic flow, thereby solving the rotation problem. Nearly all of these rely on instantaneous optic flow (i.e., a single optic flow field) (4, 9, 16–25) with few exceptions (26–29). However, it is unknown whether these algorithms are commensurate with the neural computations underlying heading perception.The consensus of opinion in the experimental literature is that human observers can estimate heading (30, 31) from instantaneous optic flow, in the absence of additional information (5, 10, 15, 32–34). Even so, there are reports of systematic biases in heading perception (11); the visual consequences of rotation (eye, head, and body) can bias heading judgments (10, 15, 35–37), with the amount of bias typically proportional to the magnitude of rotation. Other visual factors, such as stereo cues (38, 39), depth structure (8, 10, 40–43), and field of view (FOV) (33, 42–44) can modulate the strength of these biases. Errors in heading judgments have been reported to be greater when eye (35–37, 45, 46) or head movements (37) are simulated versus when they are real, which has been taken to mean that observers require extraretinal information, although there is also evidence to the contrary (10, 15, 33, 40, 41, 44, 47–50). Regardless, to date no one has tested whether heading perception (even with these biases) is based on instantaneous optic flow or on the information available in how the optic flow field evolves over time. Some have suggested that heading estimates rely on information accumulated over time (32, 44, 51), but no one has investigated the role of time-varying optic flow without confounding it with stimulus duration (i.e., the duration of evidence accumulation).In this study, we employed an application of an image processing technique that ensured that only a single optic flow field was available to observers, even though the stimulus was presented for an extended period of time. We called this condition “nonvarying phase motion” or “nonvarying”: The phases of two component gratings comprising each stationary stimulus patch shifted over time at a constant rate, causing a percept of motion in the absence of veridical movement (52). Phase motion also eliminated other cues that may otherwise have been used for heading judgments, including image point trajectories (15, 32) and their spatial compositions (i.e., looming) (53, 54). For nonvarying phase motion, observers exhibited large biases in heading judgments in the presence of rotation. A second condition, “time-varying phase motion,” or “time-varying,” included acceleration by varying the velocity of phase motion over time to match the evolution of a sequence of optic flow fields. Doing so allowed observers to compensate for the confounding effect of rotation on optic flow, making heading perception nearly veridical. This demonstrates that heading perception in the presence of rotation relies on the time-varying evolution of optic flow.     

A source for awareness-dependent figure–ground segregation in human prefrontal cortex

Figure–ground modulation, i.e., the enhancement of neuronal responses evoked by the figure relative to the background, has three complementary components: edge modulation (boundary detection), center modulation (region filling), and background modulation (background suppression). However, the neuronal mechanisms mediating these three modulations and how they depend on awareness remain unclear. For each modulation, we compared both the cueing effect produced in a Posner paradigm and fMRI blood oxygen-level dependent (BOLD) signal in primary visual cortex (V1) evoked by visible relative to invisible orientation-defined figures. We found that edge modulation was independent of awareness, whereas both center and background modulations were strongly modulated by awareness, with greater modulations in the visible than the invisible condition. Effective-connectivity analysis further showed that the awareness-dependent region-filling and background-suppression processes in V1 were not derived through intracortical interactions within V1, but rather by feedback from the frontal eye field (FEF) and dorsolateral prefrontal cortex (DLPFC), respectively. These results indicate a source for an awareness-dependent figure–ground segregation in human prefrontal cortex.

Figure–ground segregation is a fundamental process by which the visual system segments images into figures and background (1, 2). Previous neurophysiological and brain imaging studies of figure–ground segregation have shown neuronal responses are enhanced in the region perceived to be the figure and suppressed in the region perceived to be the background, an effect known as figure–ground modulation (1, 3–9). Figure–ground modulation plays a key role in identifying and localizing visual objects (7) and capturing focused attention (10). Remarkably, evidence from numerous neurophysiological (5, 11–16), psychophysical (17, 18), and brain imaging (19, 20) studies, as well as computational models (2, 17, 21) have suggested that figure–ground modulation relies on three complementary processes: boundary detection (i.e., edge modulation), region filling (i.e., center modulation), and background suppression (i.e., background modulation). During figure–ground segregation, boundary detection is the process that detects feature discontinuities that signal boundaries between the figures and background, while region filling is the process that groups figural regions with the same (or similar) features together (17), and the background suppression is the process that inhibits homogeneous features in the background (5, 22, 23). However, little is known how these three processes depend on awareness.A number of previous studies have supported an early feedforward processing phase for the boundary-detection and later feedback-processing phases for both region filling and background suppression in figure–ground modulation (2, 5, 21). These findings thus suggest that boundary detection is independent of awareness, whereas both region filling and background suppression are strongly modulated by awareness (12). Specifically, boundary detection is thought to be achieved through iso-feature inhibition (2, 21, 24, 25) within early visual areas, as early as the primary visual cortex (V1) (9, 14, 26–30), in which neurons preferring the same or similar features are more likely to suppress each other via lateral connections (31). The region-filling process, however, requires iso-feature excitation in which neurons representing the similar features enhance each other’s activity. In contrast to several studies suggesting the existence of the region-filling process within V1 (1, 9), most previous studies indicate that the region-filling process arises from feedback projections to V1 from a higher cortical area(s) (11, 12, 14, 15, 20, 32). Similarly, several neurophysiological studies have suggested that the background-suppression process may also be derived by feedback to V1 from a higher cortical area(s); it is the later processing phase in which neural activity elicited by the background is suppressed by the preceding segregated figures (5, 22, 23). However, it remains unknown which and how the top-down feedback from a higher cortical area(s) drive the region-filling and background-suppression processes in V1 that enhances the response of neurons tuned to the same feature and suppresses the neural activity elicited by the background, respectively. Also, it is unknown whether and how these feedback processes interact with awareness.Furthermore, another unclear but related issue is how boundary detection and region filling in figure–ground segregation attract focused attention. In fact, it is well known that successful segregation of a figure, as defined by an orientation contrast (Fig. 1A) from the background, leads to pop-out, which automatically attracts bottom-up attention to this salient figure location (10, 21, 25). However, it is not known whether there are different neural mechanisms by which the boundary detection and region filling attract our focused attention and whether the attentional attraction triggered by these two processes interacts with awareness.Open in a separate windowFig. 1.Stimuli, psychophysical protocol, and data. (A) Two sample orientation-defined figures presented in the upper visual field (Left: large figure; Right: small figure). The orientation contrasts between the figure bars and the background bars was 60° (the yellow dot indicates the fixation point). (B) Large (Left) and small (Right) grating probes, with the same diameter as the large and small figures, respectively. (C) Low- (Left) and high- (Right) luminance mask stimuli used in the visible and invisible conditions, respectively. (D) Psychophysical protocol. A figure–ground stimulus was presented for 50 ms, followed by a 100-ms mask and another 50-ms fixation interval. Then a large or small grating probe, with the same diameter as the large figure and small figure, respectively, was randomly presented for 50 ms with equal probability and presented randomly at either the figure location (valid cue condition) or its contralateral counterpart (invalid cue condition) with equal probability. The grating probe was orientated at 45° or 135° away from the vertical. Subjects were asked to press one of two buttons as rapidly and correctly as possible to indicate the orientation of the grating probe (45° or 135°). The psychophysical cueing effect for the large (E) and small (F) figures and the large and small gratings in both visible and invisible conditions. Each cueing effect was quantified as the difference between the reaction time of the probe task performance in the invalid cue condition and that in the valid cue condition. Error bars denote 1 SEM calculated across subjects and colored dots denote the data from each subject.To address these questions, we used a modified version of the Posner paradigm (33, 34) to measure the spatial cueing effect induced by figure boundary (edge modulation) or figure center (center modulation) of the large figure (Fig. 1 A, Left), and by the whole small figure or its surround background (background modulation, Fig. 1 A, Right). Blood oxygen level-dependent (BOLD) signals evoked by the figure boundary and figure center of the large figure, as well as the whole small figure and its surround background were also measured. Using a backward masking paradigm (10) with low- or high-luminance masks to render the whole figure–ground stimulus visible or invisible (confirmed by a two-alternative forced choice [2AFC]) to subjects, respectively, we examined how figure–ground segregation interacts with awareness. We also performed interregional correlation and effective connectivity analyses to examine the neural mechanisms of this potential interaction.     

Cingulo-opercular control network and disused motor circuits joined in standby mode

Whole-brain resting-state functional MRI (rs-fMRI) during 2 wk of upper-limb casting revealed that disused motor regions became more strongly connected to the cingulo-opercular network (CON), an executive control network that includes regions of the dorsal anterior cingulate cortex (dACC) and insula. Disuse-driven increases in functional connectivity (FC) were specific to the CON and somatomotor networks and did not involve any other networks, such as the salience, frontoparietal, or default mode networks. Censoring and modeling analyses showed that FC increases during casting were mediated by large, spontaneous activity pulses that appeared in the disused motor regions and CON control regions. During limb constraint, disused motor circuits appear to enter a standby mode characterized by spontaneous activity pulses and strengthened connectivity to CON executive control regions.

Disuse is a powerful paradigm for inducing plasticity that has uncovered key organizing principles of the human brain (1–4). Monocular deprivation—prolonged covering of one eye—revealed that multiple afferent inputs can compete for representational territory in the primary visual cortex (1). Similar competition between afferents also shapes the somatomotor system. Manipulations such as peripheral nerve deafferentation, whisker trimming, and limb constraint all drive plasticity in the primary somatosensory and motor cortex (2–4). Most plasticity studies to date have used focal techniques, such as microelectrode recordings, to study local changes in brain function. As a result, little is known about how behavior and experience shape the brain-wide functional networks that support complex cognitive operations (5).The brain is composed of networks of regions that cooperate to perform specific cognitive functions (5–8). These functional networks show synchronized spontaneous activity while the brain is at rest, a phenomenon known as resting-state functional connectivity (FC) (9–11). FC can be measured noninvasively in humans using resting-state functional MRI (rs-fMRI) and has been used to parse the brain into canonical functional networks (12, 13), including visual, auditory, and somatomotor networks (14, 15); ventral and dorsal attention networks (8, 16); a default mode network with roles in internally directed cognition and episodic memory (7, 11); a salience network thought to assess the homeostatic relevance of external stimuli (17); a frontoparietal control network supporting error processing and moment-to-moment adjustments in behavior (18–20); and a cingulo-opercular control network (CON), which maintains executive control during goal-directed behavior (18, 19, 21). Each functional network likely carries out a variety of additional functions.A more recent advance in human neuroscience has been the recognition of individual variability in network organization (22–25). Most early rs-fMRI studies examined central tendencies in network organization using group-averaged FC measurements (10, 12, 13). Recent work has demonstrated that functional networks can be identified in an individual-specific manner if sufficient rs-fMRI data are acquired, an approach termed precision functional mapping (PFM) (22, 23, 26–30). PFM respects the unique functional anatomy of each person and avoids averaging together functionally distinct brain regions across individuals.We recently demonstrated that PFM can be used to follow the time course of disuse-driven plasticity in the human brain (31). Three adult participants (Nico, Ashley, and Omar) were scanned at the same time of day for 42 to 64 consecutive days (30 min of rs-fMRI per day) before, during, and after 2 wk of dominant upper-extremity casting (Fig. 1 A and B). Casting caused persistent disuse of the dominant upper extremity during daily behaviors and led to a marked loss of strength and fine motor skill in all participants. During casting, the upper-extremity regions of the left primary somatomotor cortex (L-SM1_ue) and right cerebellum (R-Cblm_ue) functionally disconnected from the remainder of the somatomotor network. Disused motor circuits also exhibited large, spontaneous pulses of activity (Fig. 1C). Disuse pulses did not occur prior to casting, started to occur frequently within 1 to 2 d of casting, and quickly waned after cast removal.Open in a separate windowFig. 1.Experimental design and spontaneous activity pulses. (A) Three participants (Nico, Ashley, and Omar) wore casts covering the entire dominant upper extremity for 2 wk. (B) Participants were scanned every day for 42 to 64 consecutive days before, during, and after casting. All scans included 30 min of resting-state functional MRI. (C) During the Cast period, disused somatomotor circuits exhibited large pulses of spontaneous activity. (C, Left) Whole-brain ANOVA showing which brain regions contained disuse-driven pulses. (C, Right) Time courses of all pulses recorded from the disused primary somatomotor cortex.Somatomotor circuits do not function in isolation. Action selection and motor control are thought to be governed by complex interactions between the somatomotor network and control networks, including the CON (18). Prior studies of disuse-driven plasticity, including our own, have focused solely on somatomotor circuits. Here, we leveraged the whole-brain coverage of rs-fMRI and the statistical power of PFM to examine disuse-driven plasticity throughout the human brain.     

Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy

Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔG). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: 1) a nonnative lipid environment, 2) a denatured state with significant secondary structure, 3) error introduced by extrapolation to zero denaturant, and 4) the requirement of globally reversible refolding. We overcame these limitations by reversibly unfolding local regions of an individual protein with mechanical force using an atomic-force-microscope assay optimized for 2 μs time resolution and 1 pN force stability. In this assay, bR was unfolded from its native bilayer into a well-defined, stretched state. To measure ΔΔG, we introduced two alanine point mutations into an 8-amino-acid region at the C-terminal end of bR’s G helix. For each, we reversibly unfolded and refolded this region hundreds of times while the rest of the protein remained folded. Our single-molecule–derived ΔΔG for mutant L223A (−2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔG for mutant V217A was 2.2-fold larger (−2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val²¹⁷ and a natively bound squalene lipid, highlighting the contribution of membrane protein–lipid contacts not present in chemical denaturation assays. More generally, we established a platform for determining ΔΔG for a fully folded membrane protein embedded in its native bilayer.

Membrane proteins play a critical role in metabolism, transport, and signaling. Membrane proteins are also of intense biomedical interest, as they are the target for ∼50% of approved drugs (1). X-ray crystallography (2) and, more recently, cryo-electron microscopy (3–5) are yielding an accelerating number of high-resolution structures. Yet, predicting the folding and dynamics of membrane proteins remains an unmet need, in part, because it remains difficult to characterize the energetics that drive and stabilize a membrane protein’s folded structure (6–9). Membrane protein energetics are characterized in terms of the unfolding free energy (ΔG) and the change in that free energy upon introduction of a single amino acid point mutation (ΔΔG). Ideally, ΔG is interpretable as the sum of stabilizing interactions in the protein (e.g., van der Waals, hydrogen bonding), and ΔΔG isolates the energetic contribution of a single amino acid side chain to that stability. However, such an interpretation is only possible if the measurement technique reports the underlying molecular energetics with fidelity. For example, a measurement that is not made in the native bilayer may fail to accurately report stabilizing amino acid–lipid interactions. Here, we establish a single-molecule platform for measuring ΔΔG in bacteriorhodopsin (bR), a model membrane protein. The method is based upon mechanical unfolding of individual molecules in their native lipid bilayer (Fig. 1A), avoiding the nonnative detergent environment and the perturbative chemical denaturant used in traditional ensemble biochemical assays (Fig. 1B).Open in a separate windowFig. 1.Membrane protein energetics assays. (A) Illustration of the single-molecule mechanical unfolding assay where bR in its native bilayer is deposited onto a mica surface. An AFM cantilever then applies tension to the C-terminal tail, causing an eight-aa region (cyan) to reversibly unfold to a taut unfolded state (Inset). Unfolding is measured by a change in the polypeptide extension x. (B) Illustration of the traditional chemical denaturation assay showing reconstituted bR in a mixed micelle. Application of SDS then leads to a denatured state that retains ∼60% of its secondary α-helical structure (cylinders).Chemical denaturation of at least 85 bR point mutants (10–18) helps form the foundation for the current understanding of membrane protein energetics (8, 19) despite several key limitations. These measurements typically begin with reconstituted bR folded in a nonnative mixed micelle of phospholipid and detergent. Introduction of sodium dodecyl sulfate (SDS) then reversibly denatures the protein in a single step, disrupting the tertiary structure but leaving a significant fraction of secondary structure intact [∼60% as measured by circular dichroism (20)] (Fig. 1B). Analysis of the denaturant concentration dependence yields ΔG and, when repeated for point mutants, ΔΔG (21). However, this widely applied technique has four underlying limitations that can bias measured values. First, the bR is solvated in a nonnative mixed micelle, causing the free energy of the folded state to lack the energetic contributions from native protein–lipid interactions (22). Second, the measurements involve extrapolation from high denaturant concentration to zero, which assumes a linearity that steric-trapping experiments have called into question (23). Third, the free energy of the denatured state includes contributions from the ∼60% residual secondary structure (20, 24) and from nonspecific interactions with the denaturant (25). Fourth, the vast majority of membrane proteins—including all G-protein–coupled receptors—are not amenable to these measurements because there is no currently known condition under which their chemical denaturation is reversible (26).Force-induced mechanical unfolding represents an alternative means for measuring ΔΔG without the underlying limitations of chemical denaturation assays. In these atomic force microscopy (AFM) studies, the protein is initially folded in a lipid bilayer—native purple membrane in the case of bR—providing sensitivity to protein–lipid and quaternary interactions (27, 28). The cantilever of an AFM then pulls on one end of an individual membrane protein, causing it to unfold under force (F) to a stretched state (i.e., a highly extended polypeptide chain). This taut, unfolded state emerges from the bilayer into the surrounding buffer and thus contains no energetic contributions from residual secondary structure (29) or detergent interactions (Fig. 1A). As a result, both the folded and unfolded states are thermodynamically well defined, in contrast to the “unfolded” state induced by SDS denaturation (Fig. 1B). When probed with sufficient sensitivity, the unfolding process is shown to occur via a multitude of discrete states (30), each corresponding to a metastable unfolding intermediate. Reversible transitions between these states—some separated by the folding of as few as two to three amino acids (aa) (30, 31)—allow for local equilibrium energetic measurements of proteins that do not reversibly refold on larger scales.Many studies have reported AFM-based mechanical unfolding of bR (27, 30–40), including some reporting unfolding free energies (41–43); however, most prior studies lacked the ability to observe reversible transitions in the segments of bR that unfold first. In particular, assays that rely on nonspecific tip-sample attachment suffer from surface adhesion that prevents precise characterization of the first two of bR’s seven transmembrane helices to be extracted. These assays therefore cannot quantify the energetics of bR when the most native set of interactions are present. Nonspecific attachment is also not mechanically robust, preventing repeated unfolding and refolding over an extended period. Additionally, most prior AFM measurements of bR were far from equilibrium, did not account for the anomalous work of stretching the unfolded polypeptide, and had insufficient spatiotemporal resolution to observe occupancies of short-lived, closely spaced unfolding intermediates.Recent technological and methodological improvements have overcome the limitations of prior AFM-based force spectroscopy assays to allow the energetics of bR to be measured on the scale of a few aa. In particular, Yu et al. (31) showed that copper-free click chemistry can be used to form, in situ, a site-specific covalent bond between bR and an AFM tip. This chemistry allows for longer data collection and for smaller surface-contact forces, the latter reducing adhesion and enabling interpretation of the first eight aa to unfold. Additionally, Edwards et al. (44) developed focused ion beam (FIB)-modified cantilevers that provided a 100-fold improvement in time resolution and 10-fold improvement in force precision over prior studies of bR (27, 28). Recently, we combined these techniques to characterize the unfolding and refolding of the first eight aa at the C terminus of wild-type (wt) bR’s helix G, while the rest of the protein remained folded in native purple membrane. From these data, we deduced values of ΔG for these residues (∼2 kcal/mol per aa) that were consistent between three distinct analyses using both equilibrium and near-equilibrium protocols (45). Notably, this ΔG is ∼20-fold larger (on a per aa basis) than a prior chemical denaturation measurement (16). We attributed this difference in ΔG to the differing unfolded states between the two assays, with the SDS-denaturation measurement not accounting for the energy needed to disrupt the full α-helical secondary structure of bR and to solvate the unfolded protein into water (Fig. 1). Given this large difference in ΔG, it remained an open question whether consistent values of ΔΔG could be obtained between the two assays.Here, we applied these recently developed AFM techniques to measure ΔΔG of two alanine point mutants (L223A and V217A) that were previously characterized using SDS denaturation (16) and that probed different side-chain environments. Leu²²³ is oriented into the protein core of bR, whereas Val²¹⁷ is orientated outwards toward the lipid environment of purple membrane and contributes to the binding pocket of a crystallographically resolved squalene lipid (46). Ideally, ΔΔG values would agree between AFM unfolding and SDS denaturation, as both are meant to reflect the underlying contribution of the mutated side chain to the overall protein stability. Interestingly, our ΔΔG value for L223A agreed quantitatively with prior SDS-denaturation experiments despite the vastly different ΔG for the two assays. However, the AFM-based determination of ΔΔG for V217A was 2.2-fold larger than the SDS-based value, highlighting the strength of lipid–protein interactions and the importance of characterizing membrane protein energetics in the native bilayer. These measurements, therefore, constitute a practical demonstration of a widely applicable, single-molecule technique for measuring membrane protein thermodynamics that is not subject to the limitations of chemical denaturation experiments.     

From the Cover: Transgenic cotton and sterile insect releases synergize eradication of pink bollworm a century after it invaded the United States

Invasive organisms pose a global threat and are exceptionally difficult to eradicate after they become abundant in their new habitats. We report a successful multitactic strategy for combating the pink bollworm (Pectinophora gossypiella), one of the world’s most invasive pests. A coordinated program in the southwestern United States and northern Mexico included releases of billions of sterile pink bollworm moths from airplanes and planting of cotton engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). An analysis of computer simulations and 21 y of field data from Arizona demonstrate that the transgenic Bt cotton and sterile insect releases interacted synergistically to reduce the pest’s population size. In Arizona, the program started in 2006 and decreased the pest’s estimated statewide population size from over 2 billion in 2005 to zero in 2013. Complementary regional efforts eradicated this pest throughout the cotton-growing areas of the continental United States and northern Mexico a century after it had invaded both countries. The removal of this pest saved farmers in the United States $192 million from 2014 to 2019. It also eliminated the environmental and safety hazards associated with insecticide sprays that had previously targeted the pink bollworm and facilitated an 82% reduction in insecticides used against all cotton pests in Arizona. The economic and social benefits achieved demonstrate the advantages of using agricultural biotechnology in concert with classical pest control tactics.

Invasive life forms pose a major global threat and are especially difficult to eradicate after they become widespread and abundant in their new habitats (1–4). The pink bollworm (Pectinophora gossypiella), one of the world’s most invasive insects, is a voracious lepidopteran pest of cotton that was first detected in the United States in 1917 (5–8). For most of the past century, it was particularly destructive in the southwestern United States, including Arizona, where its larvae fed almost exclusively on cotton, consuming the seeds inside bolls and disrupting lint production (6, 8). In 1969, its peak seasonal density at an Arizona study site was 1.8 million larvae per hectare (ha), which translates to over 200 billion larvae in the 126,000 ha of cotton planted statewide that year (9, 10). In 1990, this pest cost Arizona cotton growers $48 million, including $32 million damage to cotton despite $16 million spent for insecticides sprayed to control it (11). In several field trials, mass releases of sterile pink bollworm moths to mate with wild moths reduced progeny production somewhat, yet did not suppress established populations because the sterile moths did not sufficiently outnumber the wild moths (6, 12–14).Pink bollworm control was revolutionized in 1996 by the introduction of cotton genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). Bt proteins kill some major insect pests yet are not toxic to most nontarget organisms, including people and many beneficial insects (15–17). Transgenic Bt cotton helped to reduce the total annual cost of pink bollworm damage and insecticide treatments to $32 million in the United States (18). Although Bt cotton kills essentially 100% of susceptible pink bollworm larvae (19–21), this pest rapidly evolved resistance to Bt proteins in laboratory selection experiments in Arizona and in Bt cotton fields in India (20–24). To delay the evolution of resistance to Bt cotton, farmers in Arizona planted “refuges” of non-Bt cotton that yielded abundant susceptible moths to mate with the rare resistant moths emerging from Bt cotton (Fig. 1A). The refuge strategy, which has been mandated in the United States and many other countries, but was not adopted widely by farmers in India, helped preserve pink bollworm susceptibility to Bt cotton in Arizona from 1996 to 2005 (24).Open in a separate windowFig. 1.Management strategies. (A) The refuge strategy is the primary approach adopted worldwide to delay the evolution of pest resistance to Bt crops and was used in Arizona from 1996 to 2005. Refuges of non-Bt cotton planted near Bt cotton produce abundant susceptible moths (blue) to mate with the rare resistant moths (red) emerging from Bt cotton. If the inheritance of resistance to Bt cotton is recessive, as in pink bollworm, the heterozygous offspring from matings between resistant and susceptible moths die when they feed on Bt cotton bolls as larvae (24). (B) Bt cotton and sterile moth releases were used together in Arizona from 2006 to 2014 as part of a multitactic program to eradicate the pink bollworm. Susceptible sterile moths (brown) were released from airplanes to mate with the rare resistant moths emerging from Bt cotton. The few progeny produced by such matings (48) are expected to be heterozygous for resistance and to die when they feed on Bt cotton bolls as larvae.As part of a coordinated, multitactic effort to eradicate the pink bollworm from the southwestern United States and northern Mexico, a new strategy largely replacing refuges with mass releases of sterile pink bollworm moths was initiated in Arizona during 2006 (Fig. 1B; 24–27). To enable this novel strategy, the US Environmental Protection Agency granted a special exemption from the refuge requirement, which allowed Arizona cotton growers to plant up to 100% of their cotton with Bt cotton (28). We previously reported data from 1998 to 2009 showing that this innovative strategy sustained susceptibility of pink bollworm to Bt cotton while reducing the pest’s population density (25). Here, to test the idea of eradicating pink bollworm with the combination of Bt cotton and sterile releases, we conducted computer simulations and analyzed field data collected in Arizona from 1998 to 2018.