Identifying hydrophobic protein patches to inform protein interaction interfaces

Interactions between proteins lie at the heart of numerous biological processes and are essential for the proper functioning of the cell. Although the importance of hydrophobic residues in driving protein interactions is universally accepted, a characterization of protein hydrophobicity, which informs its interactions, has remained elusive. The challenge lies in capturing the collective response of the protein hydration waters to the nanoscale chemical and topographical protein patterns, which determine protein hydrophobicity. To address this challenge, here, we employ specialized molecular simulations wherein water molecules are systematically displaced from the protein hydration shell; by identifying protein regions that relinquish their waters more readily than others, we are then able to uncover the most hydrophobic protein patches. Surprisingly, such patches contain a large fraction of polar/charged atoms and have chemical compositions that are similar to the more hydrophilic protein patches. Importantly, we also find a striking correspondence between the most hydrophobic protein patches and regions that mediate protein interactions. Our work thus establishes a computational framework for characterizing the emergent hydrophobicity of amphiphilic solutes, such as proteins, which display nanoscale heterogeneity, and for uncovering their interaction interfaces.

Protein–protein interactions play a crucial role in numerous biological processes, ranging from signal transduction and immune response to protein aggregation and phase behavior (1–3). Consequently, being able to understand, predict, and modulate protein interactions has important implications for understanding cellular processes and mitigating the progression of disease (4, 5). A necessary first step toward this ambitious goal is uncovering the interfaces through which proteins interact (6–8). In principle, identifying hydrophobic protein regions, which interact weakly with water, should be a promising strategy for uncovering protein interaction interfaces (9, 10). Indeed, the release of weakly interacting hydration waters from hydrophobic regions can drive protein interactions, as well as other aqueous assemblies (11–13). However, even when the structure of a protein is available at atomistic resolution, it is challenging to identify its hydrophobic patches because they are not uniformly nonpolar, but display variations in polarity and charge at the nanoscale. Moreover, the emergent hydrophobicity of a protein patch stems from the collective response of protein hydration waters to the nanoscale chemical and topographical patterns displayed by the patch (14–20) and cannot be captured by simply counting the number of nonpolar groups in the patch, or even through more involved additive approaches, such as hydropathy scales or surface-area models (21–28).To address this challenge, we build upon seminal theoretical advances and molecular simulation studies, which have shown that near a hydrophobic surface, it is easier to disrupt surface–water interactions and form interfacial cavities (29–34). To uncover protein regions that have the weakest interactions with water, here, we employ specialized molecular simulations, wherein protein–water interactions are disrupted by systematically displacing water molecules from the protein hydration shell (35–37). By identifying the protein patches that nucleate cavities most readily in our simulations, we are then able to uncover the most hydrophobic protein regions. Interestingly, we find that both hydrophobic and hydrophilic protein patches are highly heterogeneous and contain comparable numbers of nonpolar and polar atoms. Our results thus highlight the nontrivial relationship between the chemical composition of protein patches and their emergent hydrophobicity (24–26), and further emphasize the importance of accounting for the collective solvent response in characterizing protein hydrophobicity (16). We then interrogate whether the most hydrophobic protein patches, which nucleate cavities readily, are also likely to mediate protein interactions. Across five proteins that participate in either homodimer or heterodimer formation, we find that roughly 60 to 70% of interfacial contacts and only about 10 to 20% of noncontacts nucleate cavities. Our work thus provides a versatile computational framework for characterizing hydrophobicity and uncovering interaction interfaces of not just proteins, but also of other complex amphiphilic solutes, such as cavitands, dendrimers, and patchy nanoparticles (38–41).     

Self-shaping liquid crystal droplets by balancing bulk elasticity and interfacial tension

The shape diversity and controlled reconfigurability of closed surfaces and filamentous structures, universally found in cellular colonies and living tissues, are challenging to reproduce. Here, we demonstrate a method for the self-shaping of liquid crystal (LC) droplets into anisotropic and three-dimensional superstructures, such as LC fibers, LC helices, and differently shaped LC vesicles. The method is based on two surfactants: one dissolved in the LC dispersed phase and the other in the aqueous continuous phase. We use thermal stimuli to tune the bulk LC elasticity and interfacial energy, thereby transforming an emulsion of polydispersed, spherical nematic droplets into numerous, uniform-diameter fibers with multiple branches and vice versa. Furthermore, when the nematic LC is cooled to the smectic-A LC phase, we produce monodispersed microdroplets with a tunable diameter dictated by the cooling rate. Utilizing this temperature-controlled self-shaping of LCs, we demonstrate life-like smectic LC vesicle structures analogous to the biomembranes in living systems. Our experimental findings are supported by a theoretical model of equilibrium interface shapes. The shape transformation is induced by negative interfacial energy, which promotes a spontaneous increase of the interfacial area at a fixed LC volume. The method was successfully applied to many different LC materials and phases, demonstrating a universal mechanism for shape transformation in complex fluids.

Interfacial tension plays a central role in the phase separation of binary mixtures into superstructures, such as droplet emulsions, filaments, and more complex objects. It is of profound importance for an understanding of structure formation on all the length scales of complex fluids, which is crucial for the mechanical characteristics, light–matter interactions, and the advancement of the physical and chemical properties of liquids (1). The manipulation of the spherical shape of a droplet of an isotropic liquid into an elongated filament or other complex shapes is possible with the use of external fields (2), cooling (3), spontaneous emulsification (4, 5), interfacial freezing (6), and dissolution (7). Greater shape variability can be achieved in systems with complex curvature-dependent membrane elasticity, which is exploited well in intracellular biological matter, vesicles, and organelles (8–11). The interfacial effects of the membrane complement the active dynamics of the cytoskeleton to produce the characteristic shapes of different types of cells, as well as self-propulsion (12), self-division (13), chemotaxis (14), and active mass transport across the membrane, which are the key attributes of a living cell (15). In anisotropic materials, most notably liquid crystals (LCs) (16), interfacial effects are employed for the formation of droplets, shells, and topological defect structures with complex properties derived from bulk elasticity and topology (17–20), which can be used for the design of smart materials that respond to selected external stimuli (21).The self-organizing nature of anisotropic LC mesogens results in birefringent materials with long-range orientational order in the nematic (N) phase and an additional one-dimensional positional order in the smectic-A (SmA) phase (16). The average direction of an LC’s molecular alignment, defined as the director n, acts as an optical axis, making LCs suitable for various photonic applications (22–25). Variation of the orientational order in the bulk gives rise to a change in the elasticity, described by the elastic constants pertaining to the splay (K₁₁), twist (K₂₂), and bend (K₃₃) deformation modes. Controlled director deformation with external fields gives the extra advantage of the wide tunability of LC droplets’ optical properties that give them the potential to serve as soft photonic elements [i.e., waveguides, microresonators, and microlasers (26–28)] for which precise control over the size and shape is crucial. For example, smectic fibers grown in aqueous surfactant solutions prove to be excellent light guides for photonic applications (29).Over two decades ago, spontaneous filament formation was reported in a number of partially ordered liquids, specifically in binary mixtures of thermotropic LCs, which form the SmA phase while cooling from the isotropic phase or through the coexistence region of two phases (30–34). In these cases, the volume of the filaments is not conserved, which contrasts with our experiments, in which the volume of the LC is conserved and only a shape transformation takes place. Later, specially designed amphitropic surfactants were shown to transform a droplet of LC into an LC fiber during fast cooling to below the Krafft temperature of the surfactant (35). The N filaments are, in this case, transient and unstable structures, which collapse back to droplets at equilibrium. Recently, it was reported that LC polydisperse oligomers, dispersed in water containing a surfactant, form dendritic N filaments (36) and specially designed fluorinated LCs form smectic filaments (37) while cooling. These reported systems require increasingly advanced approaches and special LC materials to control the growth of the filamentous structures and to bypass the shortcomings in the control, stability, and reversibility of filamentary structures, which are in most cases structurally unstable or appear only in a narrow temperature range.In this study, we develop and systematically analyze a simple method for the creation and reversible transformation of multiple N and smectic superstructures in an aqueous host liquid. The key novelty of our method is the use of two different surfactants: one is dissolved in a droplet of LC and the other is dissolved in the water surrounding the LC droplet. The method is generic and works with very different, readily available thermotropic LCs and surfactants, which allows us a free choice of material for a desired application. We explore the interplay between the orientational elasticity of LCs and the surfactant-affected interfacial tension that leads to the shape transformation of a spherical LC droplet into single and branched fibers of uniform thickness. In addition, we form monodisperse droplets via the jet instability of the fiber driven by elastic constants of the LC and the change of the topological constraints across the phase transition into the SmA phase. Finally, we show how different life-like vesicles are spontaneously self-shaped from a single droplet in the SmA* phase and the tranformation of non-chiral SmC droplet into a helical fiber.     

Shortened tethering filaments stabilize presynaptic vesicles in support of elevated release probability during LTP in rat hippocampus

Long-term potentiation (LTP) is a cellular mechanism of learning and memory that results in a sustained increase in the probability of vesicular release of neurotransmitter. However, previous work in hippocampal area CA1 of the adult rat revealed that the total number of vesicles per synapse decreases following LTP, seemingly inconsistent with the elevated release probability. Here, electron-microscopic tomography (EMT) was used to assess whether changes in vesicle density or structure of vesicle tethering filaments at the active zone might explain the enhanced release probability following LTP. The spatial relationship of vesicles to the active zone varies with functional status. Tightly docked vesicles contact the presynaptic membrane, have partially formed SNARE complexes, and are primed for release of neurotransmitter upon the next action potential. Loosely docked vesicles are located within 8 nm of the presynaptic membrane where SNARE complexes begin to form. Nondocked vesicles comprise recycling and reserve pools. Vesicles are tethered to the active zone via filaments composed of molecules engaged in docking and release processes. The density of tightly docked vesicles was increased 2 h following LTP compared to control stimulation, whereas the densities of loosely docked or nondocked vesicles congregating within 45 nm above the active zones were unchanged. The tethering filaments on all vesicles were shorter and their attachment sites shifted closer to the active zone. These findings suggest that tethering filaments stabilize more vesicles in the primed state. Such changes would facilitate the long-lasting increase in release probability following LTP.

Long-term potentiation (LTP) is the persistent strengthening of synapses after a brief high-frequency stimulation and is widely accepted as a cellular correlate of learning and memory (1, 2). Within minutes after the induction of LTP, new receptors are inserted into the postsynaptic membrane. The resulting increase in the excitatory postsynaptic potential is immediate and can persist for hours in vitro or days to months in vivo (1, 3–8). Quantal content is also increased soon after LTP induction and reflects an increase in the number of presynaptic vesicles that release neurotransmitter (9–13). This increase in release probability is sustained several hours following LTP (14), concurrent with postsynaptic growth and spine enlargement (15). One might expect that the enhanced probability of release would involve increasing the number of vesicles docked and primed for neurotransmitter release. However, 2 h after induction of LTP, the total number of both docked and nondocked vesicles per presynaptic bouton are markedly decreased relative to control stimulation (16). These findings raise the question of whether an altered structure of docking and priming molecules leads to local clustering of vesicles that would elevate the probability of release following LTP.The proteins that connect synaptic vesicles to the plasma membrane can be visualized as filaments with electron-microscopic tomography (EMT) connecting vesicles to the presynaptic active zone (17–24). Studies suggest that the SNARE complex begins to form when a vesicle and presynaptic membrane are within 8 nm of each other rendering them loosely docked (25–27). Vesicles are then drawn toward the active zone, and the SNARE complex bundle is fully formed when the vesicle is within 2 nm of the presynaptic membrane (26, 28). Tightly docked vesicles are defined as being in contact with the presynaptic membrane and correspond to primed vesicles that comprise the readily releasable pool (27). Recent studies have suggested that docked vesicles can oscillate between loosely and tightly docked states (27, 29), providing a target mechanism for synaptic plasticity.To address the question of how changes in vesicle proximity and tethering might enhance the probability of release, we used EMT, which enabled us to acquire high-resolution structural data from small volumes of presynaptic boutons that were enriched in synaptic vesicles. We targeted active zones of hippocampal synapses, comparing their structure 2 h after LTP induction to control stimulation. The vesicle density and tethering filament dimensions were unchanged for the loosely docked and nondocked vesicles. In contrast, the density of tightly docked vesicles was increased, their tethering filaments were shorter, and the filament attachment sites on the vesicles were positioned closer to the side of the vesicle membrane facing the presynaptic membrane. Such alterations could contribute to the sustained increase in the probability of neurotransmitter release following LTP.     

Circular swimming motility and disordered hyperuniform state in an algae system

Active matter comprises individually driven units that convert locally stored energy into mechanical motion. Interactions between driven units lead to a variety of nonequilibrium collective phenomena in active matter. One of such phenomena is anomalously large density fluctuations, which have been observed in both experiments and theories. Here we show that, on the contrary, density fluctuations in active matter can also be greatly suppressed. Our experiments are carried out with marine algae (Effrenium voratum), which swim in circles at the air–liquid interfaces with two different eukaryotic flagella. Cell swimming generates fluid flow that leads to effective repulsions between cells in the far field. The long-range nature of such repulsive interactions suppresses density fluctuations and generates disordered hyperuniform states under a wide range of density conditions. Emergence of hyperuniformity and associated scaling exponent are quantitatively reproduced in a numerical model whose main ingredients are effective hydrodynamic interactions and uncorrelated random cell motion. Our results demonstrate the existence of disordered hyperuniform states in active matter and suggest the possibility of using hydrodynamic flow for self-assembly in active matter.

Active matter exists over a wide range of spatial and temporal scales (1–6) from animal groups (7, 8) to robot swarms (9–11), to cell colonies and tissues (12–16), to cytoskeletal extracts (17–20), to man-made microswimmers (21–25). Constituent particles in active matter systems are driven out of thermal equilibrium at the individual level; they interact to develop a wealth of intriguing collective phenomena, including clustering (13, 22, 24), flocking (11, 26), swarming (12, 13), spontaneous flow (14, 20), and giant density fluctuations (10, 11). Many of these observed phenomena have been successfully described by particle-based or continuum models (1–6), which highlight the important roles of both individual motility and interparticle interactions in determining system dynamics.Current active matter research focuses primarily on linearly swimming particles which have a symmetric body and self-propel along one of the symmetry axes. However, a perfect alignment between the propulsion direction and body axis is rarely found in reality. Deviation from such a perfect alignment leads to a persistent curvature in the microswimmer trajectories; examples of such circle microswimmers include anisotropic artificial micromotors (27, 28), self-propelled nematic droplets (29, 30), magnetotactic bacteria and Janus particles in rotating external fields (31, 32), Janus particle in viscoelastic medium (33), and sperm and bacteria near interfaces (34, 35). Chiral motility of circle microswimmers, as predicted by theoretical and numerical investigations, can lead to a range of interesting collective phenomena in circular microswimmers, including vortex structures (36, 37), localization in traps (38), enhanced flocking (39), and hyperuniform states (40). However, experimental verifications of these predictions are limited (32, 35), a situation mainly due to the scarcity of suitable experimental systems.Here we address this challenge by investigating marine algae Effrenium voratum (41, 42). At air–liquid interfaces, E. voratum cells swim in circles via two eukaryotic flagella: a transverse flagellum encircling the cellular anteroposterior axis and a longitudinal one running posteriorly. Over a wide range of densities, circling E. voratum cells self-organize into disordered hyperuniform states with suppressed density fluctuations at large length scales. Hyperuniformity (43, 44) has been considered as a new form of material order which leads to novel functionalities (45–49); it has been observed in many systems, including avian photoreceptor patterns (50), amorphous ices (51), amorphous silica (52), ultracold atoms (53), soft matter systems (54–61), and stochastic models (62–64). Our work demonstrates the existence of hyperuniformity in active matter and shows that hydrodynamic interactions can be used to construct hyperuniform states.     

Cryoelectron-microscopy structure of the enteropathogenic Escherichia coli type III secretion system EspA filament

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) utilize a macromolecular type III secretion system (T3SS) to inject effector proteins into eukaryotic cells. This apparatus spans the inner and outer bacterial membranes and includes a helical needle protruding into the extracellular space. Thus far observed only in EPEC and EHEC and not found in other pathogenic Gram-negative bacteria that have a T3SS is an additional helical filament made by the EspA protein that forms a long extension to the needle, mediating both attachment to eukaryotic cells and transport of effector proteins through the intestinal mucus layer. Here, we present the structure of the EspA filament from EPEC at 3.4 Å resolution. The structure reveals that the EspA filament is a right-handed 1-start helical assembly with a conserved lumen architecture with respect to the needle to ensure the seamless transport of unfolded cargos en route to the target cell. This functional conservation is despite the fact that there is little apparent overall conservation at the level of sequence or structure with the needle. We also unveil the molecular details of the immunodominant EspA epitope that can now be exploited for the rational design of epitope display systems.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea, morbidity, and mortality in low- and middle-income countries (1), while enterohemorrhagic E. coli (EHEC) is a major cause of food poisoning in industrial countries (2). The ability of EPEC and EHEC to colonize the intestinal epithelium is encoded on a pathogenicity island named the locus of enterocyte effacement, which encodes gene regulators, the outer membrane adhesin intimin, structural components of a type III secretion system (T3SS), translocon components, chaperones, effectors, and an ATPase, which energizes protein translocation (3).T3SS is a common virulence factor among Gram-negative pathogens of humans, animals, and plants, including Salmonella enterica serovars, Shigella, Chlamydia, and Yersinia spp., Pseudomonas aeruginosa, and Pseudomonas syringae (4). The overall architecture of the multisubunit T3SS injectisome, which spans the entire cell envelope, is highly conserved among the different pathogens. It comprises several substructures, including a cytosolic C-ring and an ATPase complex (EscN in EPEC), a basal body consisting of a series of ring structures embedded in the bacterial inner and outer membranes (including a Secretin, EscC) and a periplasmic rod (EscI) which connects the inner membrane rings with a hollow extracellular needle projection (EscF). In EPEC, the EscF needle is 8–9 nm in diameter and 23 nm in length (5–7). In most pathogenic bacteria having a T3SS, the function of the needle is to connect the basal body to a translocation pore in the plasma membrane of the eukaryotic cell (3, 4).Electron microscopic observations of EPEC and EHEC have shown an ∼12-nm-diameter helical tube, made of the secreted translocator protein EspA (8–10), which serves as an extension to the needle, enclosing a central channel of ∼25 Å diameter (11, 12). Around 12 EspA filaments are elaborated on individual EPEC bacteria (8); when grown in vitro, EspA can vary in length and can reach 600 nm (7). Our current understanding is that the EPEC and EHEC EspA filaments evolved as an adaptation to their environment, where the needle alone would not be long enough to traverse the intestinal mucus layer. EspA filaments, like the needle, share similar helical symmetry parameters with flagellar structures and are elongated by addition of EspA subunits to the tip of the growing filament, the same mode of elongation that occurs in flagella filaments (13). Functionally, the filaments form a long flexible helical conduit which connects the tip of the needle with the translocation pore (made in EPEC by EspB and EspD), thus mediating effector translocation (3). Indeed, EspA has been shown to interact with both EspB and EspD (14, 15). In addition to their protein translocation activity, EspA filaments are important adhesins, mediating binding to both epithelial cells and edible leaves (16, 17). In the absence of EspA filaments, effectors can be secreted but not translocated; accordingly, the virulence of an espA deletion is highly attenuated in animal models (18). Moreover, EspA filaments are major antigens in vivo; antibodies against EspA were found in both human colostrum of mothers in Brazil and in serum from culture-positive patients infected with EHEC (19, 20). In animal models, IgG antibodies against EspA play a major role in clearing the pathogen (21).EspA alone is sufficient to form filamentous structures (22). Similarly to flagellar biosynthesis (23), EspA coiled-coil interactions between N- and C-terminal α-helical segments are required for assembly of the filament (24, 25). Monomeric EspA subunits are maintained in the cytosol via interactions with the chaperone CesAB (26), which has also been called CesA (22). CesAB is essential for stability of EspA within the bacterial cell prior to secretion. A cesAB deletion cannot secrete EspA or assemble EspA filaments (26). Crystallographic analysis of the CesAB–EspA complex at 2.8 Å resolution (22) showed that the EspA α-helices are also involved in extensive coiled-coil interactions with CesAB (22). Due to disorder in the parts of EspA not directly interacting with CesAB, only 72 of the 192 EspA residues were visualized in this complex. Importantly, the ATPase EscN selectively interacts with the CesAB–EspA complex; abrogation of this interaction attenuates EspA secretion and infection (27).Like flagella, EspA filaments show antigenic polymorphism, as EspA from different EPEC and EHEC clones show no immunological cross-reactivity (10). We have previously identified a surface-exposed hypervariable domain that contains the immunodominant EspA epitope (28). By exchanging the hypervariable domains of EspA(EPEC) and EspA(EHEC) we swapped the antigenic specificity of the EspA filaments (26). As with the Salmonella flagellin D3 domain (29), which is known to tolerate insertions of natural and artificial amino acid sequences (30), we were able to insert short peptides into the surface-exposed, hypervariable region of EspA (28).While EspA was first identified in 1996 (31), and the EspA filaments were first described in 1998 (8), low-resolution structures (at ∼15–25 Å resolution) were reported about 15 y ago (11, 12). The aim of this study was to obtain a high-resolution cryoelectron microscopy (cryo-EM) structure of the EPEC EspA filament.     

CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left–right symmetry breaking

Proper left–right symmetry breaking is essential for animal development, and in many cases, this process is actomyosin-dependent. In Caenorhabditis elegans embryos active torque generation in the actomyosin layer promotes left–right symmetry breaking by driving chiral counterrotating cortical flows. While both Formins and Myosins have been implicated in left–right symmetry breaking and both can rotate actin filaments in vitro, it remains unclear whether active torques in the actomyosin cortex are generated by Formins, Myosins, or both. We combined the strength of C. elegans genetics with quantitative imaging and thin film, chiral active fluid theory to show that, while Non-Muscle Myosin II activity drives cortical actomyosin flows, it is permissive for chiral counterrotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counterrotation and promotes in-plane, active torque generation in the actomyosin cortex. Notably, we observe that artificially generated large active RhoA patches undergo rotations with consistent handedness in a CYK-1/Formin–dependent manner. Altogether, we conclude that CYK-1/Formin–dependent active torque generation facilitates chiral symmetry breaking of actomyosin flows and drives organismal left–right symmetry breaking in the nematode worm.

The emergence of left–right asymmetry is essential for normal animal development and, in the majority of animal species, one type of handedness is dominant (1). The actin cytoskeleton plays an instrumental role in establishing the left–right asymmetric body plan of invertebrates like fruit flies (2–6), nematodes (7–11), and pond snails (12–15). Moreover, an increasing number of studies showed that vertebrate left–right patterning also depends on a functional actomyosin cytoskeleton (13, 16–22). Actomyosin-dependent chiral behavior has even been reported in isolated cells (23–28) and such cell-intrinsic chirality has been shown to promote left–right asymmetric morphogenesis of tissues (29, 30), organs (21, 31), and entire embryonic body plans (12, 13, 32, 33). Active force generation in the actin cytoskeleton is responsible for shaping cells and tissues during embryo morphogenesis. Torques are rotational forces with a given handedness and it has been proposed that in plane, active torque generation in the actin cytoskeleton drives chiral morphogenesis (7, 8, 34, 35).What could be the molecular origin of these active torques? The actomyosin cytoskeleton consists of actin filaments, actin-binding proteins, and Myosin motors. Actin filaments are polar polymers with a right-handed helical pitch and are therefore chiral themselves (36, 37). Due to the right-handed pitch of filamentous actin, Myosin motors can rotate actin filaments along their long axis while pulling on them (33, 38–42). Similarly, when physically constrained, members of the Formin family rotate actin filaments along their long axis while elongating them (43). In both cases the handedness of this rotation is determined by the helical nature of the actin polymer. From this it follows that both Formins and Myosins are a potential source of molecular torque generation that could drive cellular and organismal chirality. Indeed, chiral processes across different length scales, and across species, are dependent on Myosins (19), Formins (13–15, 26), or both (7, 8, 21, 44). It is, however, unclear how Formins and Myosins contribute to active torque generation and the emergence chiral processes in developing embryos.In our previous work we showed that the actomyosin cortex of some Caenorhabditis elegans embryonic blastomeres undergoes chiral counterrotations with consistent handedness (7, 35). These chiral actomyosin flows can be recapitulated using active chiral fluid theory that describes the actomyosin layer as a thin-film, active gel that generates active torques (7, 45, 46). Chiral counterrotating cortical flows reorient the cell division axis, which is essential for normal left–right symmetry breaking (7, 47). Moreover, cortical counterrotations with the same handedness have been observed in Xenopus one-cell embryos (32), suggesting that chiral counterrotations are conserved among distant species. Chiral counterrotating actomyosin flow in C. elegans blastomeres is driven by RhoA signaling and is dependent on Non-Muscle Myosin II motor proteins (7). Moreover, the Formin CYK-1 has been implicated in actomyosin flow chirality during early polarization of the zygote as well as during the first cytokinesis (48, 49). Despite having identified a role for Myosins and Formins, the underlying mechanism by which active torques are generated remains elusive.Here we show that the Diaphanous-like Formin, CYK-1/Formin, is a critical determinant for the emergence of actomyosin flow chirality, while Non-Muscle Myosin II (NMY-2) plays a permissive role. Our results show that cortical CYK-1/Formin is recruited by active RhoA signaling foci and promotes active torque generation, which in turn tends to locally rotate the actomyosin cortex clockwise. In the highly connected actomyosin meshwork, a gradient of these active torques drives the emergence of chiral counterrotating cortical flows with uniform handedness, which is essential for proper left–right symmetry breaking. Together, these results provide mechanistic insight into how Formin-dependent torque generation drives cellular and organismal left–right symmetry breaking.     

Improved bounds on entropy production in living systems

Living systems maintain or increase local order by working against the second law of thermodynamics. Thermodynamic consistency is restored as they consume free energy, thereby increasing the net entropy of their environment. Recently introduced estimators for the entropy production rate have provided major insights into the efficiency of important cellular processes. In experiments, however, many degrees of freedom typically remain hidden to the observer, and, in these cases, existing methods are not optimal. Here, by reformulating the problem within an optimization framework, we are able to infer improved bounds on the rate of entropy production from partial measurements of biological systems. Our approach yields provably optimal estimates given certain measurable transition statistics. In contrast to prevailing methods, the improved estimator reveals nonzero entropy production rates even when nonequilibrium processes appear time symmetric and therefore may pretend to obey detailed balance. We demonstrate the broad applicability of this framework by providing improved bounds on the energy consumption rates in a diverse range of biological systems including bacterial flagella motors, growing microtubules, and calcium oscillations within human embryonic kidney cells.

Thermodynamic laws place fundamental limits on the efficiency and fitness of living systems (1, 2). To maintain cellular order and perform essential biological functions such as sensing (3–6), signaling (7), replication (8, 9) or locomotion (10), organisms consume energy and dissipate heat. In doing so, they increase the entropy of their environment (2), in agreement with the second law of thermodynamics (11). Obtaining reliable estimates for the energy consumption and entropy production in living matter holds the key to understanding the physical boundaries (12–14) that constrain the range of theoretically and practically possible biological processes (3). Recent experimental (6, 15, 16) and theoretical (17–20) advances in the imaging and modeling of cellular and subcellular dynamics have provided groundbreaking insights into the thermodynamic efficiency of molecular motors (14, 21), biochemical signaling (16, 22, 23) and reaction (24) networks, and replication (9) and adaption (25) phenomena. Despite such major progress, however, it is also known that the currently available entropy production estimators (26, 27) can fail under experimentally relevant conditions, especially when only a small set of observables is experimentally accessible or nonequilibrium transport currents (28–30) vanish.To help overcome these limitations, we introduce here a generic optimization framework that can produce significantly improved bounds on the entropy production in living systems. We will prove that these bounds are optimal given certain measurable statistics. From a practical perspective, our method requires observations of only a few coarse-grained state variables of an otherwise hidden Markovian network. We demonstrate the practical usefulness by determining improved entropy production bounds for bacterial flagella motors (10, 31), growing microtubules (32, 33), and calcium oscillations (7, 34) in human embryonic kidney cells.Generally, entropy production rates can be estimated from the time series of stochastic observables (35). Thermal equilibrium systems obey the principle of detailed balance, which means that every forward trajectory is as likely to be observed as its time reversed counterpart, neutralizing the arrow of time (36). By contrast, living organisms operate far from equilibrium, which means that the balance between forward and reversed trajectories is broken and net fluxes may arise (1, 37–39). When all microscopic details of a nonequilibrium system are known, one can measure the rate of entropy production by comparing the likelihoods of forward and reversed trajectories in sufficiently large data samples (35, 36). However, in most if not all biophysical experiments, many degrees of freedom remain hidden to the observer, demanding methods (28, 40, 41) that do not require complete knowledge of the system. A powerful alternative is provided by thermodynamic uncertainty relations (TURs), which use the mean and variance of steady-state currents to bound entropy production rates (18, 19, 26, 42–48). Although highly useful when currents can be measured (44–47), or when the system can be externally manipulated (40, 49), these methods give, by construction, trivial zero bounds for current-free nonequilibrium systems, such as driven one-dimensional (1D) nonperiodic oscillators. In the absence of currents, potential asymmetries in the forward and reverse trajectories can still be exploited to bound the entropy production rate (29, 30, 50), but to our knowledge no existing method is capable of producing nonzero bounds when forward and reverse trajectories are statistically identical. Moreover, even though previous bounds can become tight in some cases (51), optimal entropy production estimators for nonequilibrium systems are in general unknown.To obtain bounds that are provably optimal under reasonable conditions on the available data, we reformulate the problem here within an optimization framework. Formally, we consider any steady-state Markovian dynamics for which only coarse-grained variables are observable, where these observables may appear non-Markovian. We then search over all possible underlying Markovian systems to identify the one which minimizes the entropy production rate while obeying the observed statistics. More specifically, our algorithmic implementation leverages information about successive transitions, allowing us to discover nonzero bounds on entropy production even when the coarse-grained statistics appear time symmetric. We demonstrate this for both synthetic test data and experimental data (52) for flagella motors. Subsequently, we consider the entropy production of microtubules (33), which slowly grow before rapidly shrinking in steady state, to show how refined coarse graining in space and time leads to improved bounds. The final application to calcium oscillations in human embryonic kidney cells (34) illustrates how external stimulation with drugs can increase entropy production.     

From the Cover: Emergence of dynamic vortex glasses in disordered polar active fluids

In equilibrium, disorder conspires with topological defects to redefine the ordered states of matter in systems as diverse as crystals, superconductors, and liquid crystals. Far from equilibrium, however, the consequences of quenched disorder on active condensed matter remain virtually uncharted. Here, we reveal a state of strongly disordered active matter with no counterparts in equilibrium: a dynamical vortex glass. Combining microfluidic experiments and theory, we show how colloidal flocks collectively cruise through disordered environments without relaxing the topological singularities of their flows. The resulting state is highly dynamical but the flow patterns, shaped by a finite density of frozen vortices, are stationary and exponentially degenerated. Quenched isotropic disorder acts as a random gauge field turning active liquids into dynamical vortex glasses. We argue that this robust mechanism should shape the collective dynamics of a broad class of disordered active matter, from synthetic active nematics to collections of living cells exploring heterogeneous media.

From a physicist’s perspective, flocks are ensembles of living or synthetic motile units collectively moving along a common emerging direction (1–4). They realize one of the most robust ordered states of matter observed over five orders of magnitude in scale and in systems as diverse as motility assays, self-propelled colloids, shaken grains, and actual flocks of birds (3, 5–10). The quiet flows of flocks are in stark contrast with the spatiotemporal chaos consistently reported and predicted in active nematic liquid crystals, another abundant form of ordered active matter realized in biological tissues, swimming cells, cellular extracts, and shaken rods (2, 11). Active nematics do not support any form of long-range order (4, 12). Their structure is continuously bent and destroyed by the proliferation and annihilation of singularities in their local orientation: topological defects (11, 13–15). Unlike in active nematics, topological defects in flocking matter are merely transient excitations which annihilate rapidly and allow uniaxial order to extend over system-spanning scales (4).This idyllic view of the ordered phases of active liquids is limited, however, to pure systems. Disorder is known to profoundly alter the stability of topological defects and the corresponding ordered states in equilibrium condensed matter (16–18), but its role in active fluids remains virtually uncharted territory. All previous studies (19–26), including our own early experiments (22), have been limited to weak disorder and smooth perturbations around topologically trivial states. Unlike in equilibrium, no available experiment, simulation, or theory has ever demonstrated or predicted disorder-induced topological excitations in active matter.In this paper we show how isotropic disorder generically challenges the extreme robustness of flocking matter to topological defects. We map the full phase behavior of colloidal flocks navigating through disordered lattice of obstacles and reveal an unanticipated state of active matter: a dynamical vortex glass. In dynamical vortex glasses, millions of self-propelled particles can steadily cruise through disorder, maintaining local orientational order and without relaxing the topological singularities of their flows. The associated flow patterns are exponentially degenerated and shaped by amorphous ensembles of frozen topological defects, yielding a dynamical state akin to the static vortex-glass phase of dirty superconductors and random-gauge magnets (27–29). Building a theory of flock hydrodynamics beyond the spin-wave approximation, we elucidate the emergence and stabilization of topological vortices by quenched disorder. Finally, we discuss the universality of the dynamical vortex glass phase beyond the specifics of polar active matter and colloidal flocks.     

Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy

Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases and induces optic nerve regeneration in animal models. Paradoxically, however, although CNTF gene therapy promotes extensive regeneration, recombinant CNTF (rCNTF) has little effect. Because intraocular viral vectors induce inflammation, and because CNTF is an immune modulator, we investigated whether CNTF gene therapy acts indirectly through other immune mediators. The beneficial effects of CNTF gene therapy remained unchanged after deleting CNTF receptor alpha (CNTFRα) in retinal ganglion cells (RGCs), the projection neurons of the retina, but were diminished by depleting neutrophils or by genetically suppressing monocyte infiltration. CNTF gene therapy increased expression of C-C motif chemokine ligand 5 (CCL5) in immune cells and retinal glia, and recombinant CCL5 induced extensive axon regeneration. Conversely, CRISPR-mediated knockdown of the cognate receptor (CCR5) in RGCs or treating wild-type mice with a CCR5 antagonist repressed the effects of CNTF gene therapy. Thus, CCL5 is a previously unrecognized, potent activator of optic nerve regeneration and mediates many of the effects of CNTF gene therapy.

Like most pathways in the mature central nervous system (CNS), the optic nerve cannot regenerate once damaged due in part to cell-extrinsic suppressors of axon growth (1, 2) and the low intrinsic growth capacity of adult retinal ganglion cells (RGCs), the projection neurons of the eye (3–5). Consequently, traumatic or ischemic optic nerve injury or degenerative diseases such as glaucoma lead to irreversible visual losses. Experimentally, some degree of regeneration can be induced by intraocular inflammation or growth factors expressed by inflammatory cells (6–10), altering the cell-intrinsic growth potential of RGCs (3–5), enhancing physiological activity (11, 12), chelating free zinc (13, 14), and other manipulations (15–19). However, the extent of regeneration achieved to date remains modest, underlining the need for more effective therapies.Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for glaucoma and other ocular diseases (20–23). Activation of the downstream signal transduction cascade requires CNTF to bind to CNTF receptor-α (CNTFRα) (24), which leads to recruitment of glycoprotein 130 (gp130) and leukemia inhibitory factor receptor-β (LIFRβ) to form a tripartite receptor complex (25). CNTFRα anchors to the plasma membrane through a glycosylphosphatidylinositol linkage (26) and can be released and become soluble through phospholipase C-mediated cleavage (27). CNTF has been reported to activate STAT3 phosphorylation in retinal neurons, including RGCs, and to promote survival, but it is unknown whether these effects are mediated by direct action of CNTF on RGCs via CNTFRα (28). Our previous studies showed that CNTF promotes axon outgrowth from neonate RGCs in culture (29) but fails to do so in cultured mature RGCs (8) or in vivo (6). Although some studies report that recombinant CNTF (rCNTF) can promote optic nerve regeneration (20, 30, 31), others find little or no effect unless SOCS3 (suppressor of cytokine signaling-3), an inhibitor of the Jak-STAT pathway, is deleted in RGCs (5, 6, 32). In contrast, multiple studies show that adeno-associated virus (AAV)-mediated expression of CNTF in RGCs induces strong regeneration (33–40). The basis for the discrepant effects of rCNTF and CNTF gene therapy is unknown but is of considerable interest in view of the many promising clinical and preclinical outcomes obtained with CNTF to date.Because intravitreal virus injections induce inflammation (41), we investigated the possibility that CNTF, a known immune modulator (42–44), might act by elevating expression of other immune-derived factors. We report here that the beneficial effects of CNTF gene therapy in fact require immune system activation and elevation of C-C motif chemokine ligand 5 (CCL5). Depletion of neutrophils, global knockout (KO) or RGC-selective deletion of the CCL5 receptor CCR5, or a CCR5 antagonist all suppress the effects of CNTF gene therapy, whereas recombinant CCL5 (rCCL5) promotes axon regeneration and increases RGC survival. These studies point to CCL5 as a potent monotherapy for optic nerve regeneration and to the possibility that other applications of CNTF and other forms of gene therapy might similarly act indirectly through other factors.     

Evolution and global charge conservation for polarization singularities emerging from non-Hermitian degeneracies

Core concepts in singular optics, especially the polarization singularities, have rapidly penetrated the surging fields of topological and non-Hermitian photonics. For open photonic structures with non-Hermitian degeneracies in particular, polarization singularities would inevitably encounter another sweeping concept of Berry phase. Several investigations have discussed, in an inexplicit way, connections between both concepts, hinting at that nonzero topological charges for far-field polarizations on a loop are inextricably linked to its nontrivial Berry phase when degeneracies are enclosed. In this work, we reexamine the seminal photonic crystal slab that supports the fundamental two-level non-Hermitian degeneracies. Regardless of the invariance of nontrivial Berry phase (concerning near-field Bloch modes defined on the momentum torus) for different loops enclosing both degeneracies, we demonstrate that the associated far polarization fields (defined on the momentum sphere) exhibit topologically inequivalent patterns that are characterized by variant topological charges, including even the trivial scenario of zero charge. Moreover, the charge carried by the Fermi arc actually is not well defined, which could be different on opposite bands. It is further revealed that for both bands, the seemingly complex evolutions of polarizations are bounded by the global charge conservation, with extra points of circular polarizations playing indispensable roles. This indicates that although not directly associated with any local charges, the invariant Berry phase is directly linked to the globally conserved charge, physical principles underlying which have all been further clarified by a two-level Hamiltonian with an extra chirality term. Our work can potentially trigger extra explorations beyond photonics connecting Berry phase and singularities.

Pioneered by Pancharatnam, Berry, Nye, and others (1–10), Berry phase and singularities have become embedded languages all across different branches of photonics. Optical Berry phase is largely manifested through either polarization evolving Pancharatnam–Berry phase or the spin-redirection Bortolotti–Rytov–Vladimirskii–Berry phase (2, 4, 5, 11–15); while optical singularities are widely observed as singularities of intensity (caustics) (6), phase (vortices) (7), or polarization (8–10). As singularities for complex vectorial waves, polarization singularities are skeletons of electromagnetic waves and are vitally important for understanding various interference effects underlying many applications (16–20).There is a superficial similarity between the aforementioned two concepts: Both the topological charge of polarization field [Hopf index of line field (21, 22)] and Berry phase are defined on a closed circuit. Despite this, it is quite unfortunate that almost no definite connections have been established between them in optics. This is fully understandable: Berry phase is defined on the Pancharatnam connection (parallel transport) that decides the phase contrast between neighboring states on the loop (3, 4); while the polarization charge reflects accumulated orientation rotations of polarization ellipses, which has no direct relevance to the overall phase of each state. This explains why in pioneering works where both concepts were present (23–27), their interplay was rarely elaborated on.Spurred by studies into bound states in the continuum, polarization singularities have gained enormous renewed interest in open periodic photonic structures, manifested in different morphologies with both fundamental and higher-order half-integer charges (28–50). Simultaneously, the significance of Berry phase has been further reinforced in surging fields of topological and non-Hermitian photonics (1, 23, 26, 51–56). In open periodic structures involving band degeneracies, Berry phase and polarization singularity would inevitably meet, which sparks the influential work on non-Hermitian degeneracy (36) and several other following studies (40, 43, 45) discussing both concepts simultaneously. Although not claimed explicitly, those works hint that nontrivial Berry phase produces nonzero polarization charges.Aiming to bridge Berry phase and polarization singularity, we reexamine the seminal photonic crystal slab (PCS) that supports elementary two-level non-Hermitian degeneracies. It is revealed that with an invariant nontrivial π Berry phase, the corresponding polarization fields on different isofrequency contours enclosing both non-Hermitian degenerate points (or equivalently exceptional points [EPs]) (26) exhibit diverse patterns characterized by different polarization charges, even including the trivial zero charge. It is further revealed that the charge carried by the Fermi arc is actually not well defined, which could be different on opposite bands. We also discover that such complexity of field evolutions is constrained by global charge conservation for both bands, with extra points of circular polarizations (C points) playing pivotal roles. This reveals the explicit connection between globally conserved charge and the invariant Berry phase, underlying which the physical mechanisms have been further clarified by a two-level Hamiltonian with an extra chirality term (25). We show that such an unexpected connection is generically manifest in various structures, despite the fact that Berry phase and polarization charge actually characterize different entities of near-field Bloch modes and their projected far polarization fields, respectively: Bloch modes are defined on the momentum torus and can be folded into the irreducible Brillouin zone; while polarization fields are defined on the momentum sphere, due to the involvement of out-of-plane wave vectors along which there is no periodicity. Our study can spur further investigations in other subjects beyond photonics to explore conceptual interconnectedness, where both the concepts of Berry phase and singularities are present.     

Hierarchical supramolecular assembly of a single peptoid polymer into a planar nanobrush with two distinct molecular packing motifs

Hierarchical nanomaterials have received increasing interest for many applications. Here, we report a facile programmable strategy based on an embedded segmental crystallinity design to prepare unprecedented supramolecular planar nanobrush-like structures composed of two distinct molecular packing motifs, by the self-assembly of one particular diblock copolymer poly(ethylene glycol)-block-poly(N-octylglycine) in a one-pot preparation. We demonstrate that the superstructures result from the temperature-controlled hierarchical self-assembly of preformed spherical micelles by optimizing the crystallization−solvophobicity balance. Particularly remarkable is that these micelles first assemble into linear arrays at elevated temperatures, which, upon cooling, subsequently template further lateral, crystallization-driven assembly in a living manner. Addition of the diblock copolymer chains to the growing nanostructure occurs via a loosely organized micellar intermediate state, which undergoes an unfolding transition to the final crystalline state in the nanobrush. This assembly mechanism is distinct from previous crystallization-driven approaches which occur via unimer addition, and is more akin to protein crystallization. Interestingly, nanobrush formation is conserved over a variety of preparation pathways. The precise control ability over the superstructure, combined with the excellent biocompatibility of polypeptoids, offers great potential for nanomaterials inaccessible previously for a broad range of advanced applications.

Biomacromolecules fold and assemble into complex, well-organized hierarchical structures by a network of noncovalent interactions, which enable tremendous architectural diversity in nature (1, 2). For example, polypeptide chains encoded with assembly information in their monomer sequence can fold into highly ordered conformations, which give rise to their biological functionality (3). Inspired by these intricate natural designs, numerous efforts have been devoted to fabricating hierarchical nanostructures using synthetic polymeric materials (4–11). However, the precision control over the assembly process and structure across many length scales, as commonly seen in biomacromolecules, remains a challenging task (12). This is because the assembly information content encoded within synthetic macromolecules is considerably lower.Synthetic chemists have looked to develop polymer systems that retain many of the structural features found in natural biopolymers. Bioinspired synthetic polymers have attracted growing attention, because of their inherent structural advantages, facile synthetic approaches, and improved stability, to serve as promising materials for the de novo design and assembly of hierarchical nanostructures. In particular, polypeptoids are a class of peptidomimetic polymers based on a polar amide backbone (13–15). This differs substantially from carbon-chain polymers such as polyethylene and polypropylene. The amide groups impart higher water solubility, excellent biocompatibility, the opportunity for multiple backbone−backbone interactions, and enable a wide range of bioactivities. Polypeptoids are devoid of hydrogen bond donating sites and chirality on the backbone due to the N substitution. This simplifies the complex molecular interactions inherent in peptidomimetic materials, resulting in efficient engineering and controllable architecture construction. For example, polypeptoids with alkyl side chain groups are semicrystalline with inherent characteristic packing domains, which is in sharp contrast to polypeptides (16–19).Macromolecular crystallization is an essential process in both nature and materials manufacturing. The self-assembly of block copolymers containing crystalline blocks generally enables the formation of multiscale architectures with a high level of control over morphology and dimension (20, 21). Recently, living crystallization-driven self-assembly has been demonstrated to be an effective strategy to precisely control the nanoscale morphology (22–30). Inspired by the natural encoded information-guided self-assembly, we based our design on a hydrophobic poly(N-alkylglycine) peptoid block that is known to form a rectangular crystalline lattice with controllable dimension and two melting transitions (31). It is also known that solvophobic interaction is the predominant driving force for assembly of systems with solvophobic segments (5). The delicate interplay between crystallization and solvophobicity is essential for biomolecule self-assembly (32), which potentially serves as a powerful strategy for self-assembly of block copolymers. Thus, we embarked on a study of block copolymers, where the relative strength of these two factors could be systematically adjusted. By optimizing the balance between these two factors, we discovered that poly(ethylene glycol)-block-poly(N-octylglycine) (PEG-b-PNOG) formed unique supramolecular planar nanobrush architectures in high yield. We developed a simple temperature-controlled assembly strategy to create superbrushes consisting of two distinct packing domains: a long core fiber, or “spine,” with lengths up to ∼2.0 µm, and laterally splayed shorter fibers of ∼400 nm in length on apposed side surfaces of the spine. We further demonstrated that this lateral growth of the brush exhibits living growth manner via a micelle intermediate, fairly distinct from known living crystallization-driven self-assembly approaches (16, 23, 33), where assemblies grow via the direct attachment of block unimers. Our results coincide with the reported “particle attachment” strategy observed in a range of biomacromolecules and small molecules, where intermediate higher-ordered species form in solution prior to their attachment to the crystal lattice, in contrast to monomer-by-monomer crystal growth (1, 32, 34, 35). Such pathways allow for the optimization of interactions to facilitate thermodynamic favored transition from the initial species to hierarchical assemblies.     

Naphthylphthalamic acid associates with and inhibits PIN auxin transporters

N-1-naphthylphthalamic acid (NPA) is a key inhibitor of directional (polar) transport of the hormone auxin in plants. For decades, it has been a pivotal tool in elucidating the unique polar auxin transport-based processes underlying plant growth and development. Its exact mode of action has long been sought after and is still being debated, with prevailing mechanistic schemes describing only indirect connections between NPA and the main transporters responsible for directional transport, namely PIN auxin exporters. Here we present data supporting a model in which NPA associates with PINs in a more direct manner than hitherto postulated. We show that NPA inhibits PIN activity in a heterologous oocyte system and that expression of NPA-sensitive PINs in plant, yeast, and oocyte membranes leads to specific saturable NPA binding. We thus propose that PINs are a bona fide NPA target. This offers a straightforward molecular basis for NPA inhibition of PIN-dependent auxin transport and a logical parsimonious explanation for the known physiological effects of NPA on plant growth, as well as an alternative hypothesis to interpret past and future results. We also introduce PIN dimerization and describe an effect of NPA on this, suggesting that NPA binding could be exploited to gain insights into structural aspects of PINs related to their transport mechanism.

Many aspects of plant growth are controlled by the hormone auxin. A distinct feature of auxin is that its hormonal action requires it to be actively transported between cells and ultimately throughout the whole plant in a controlled directional or polarized manner, a process known as polar auxin transport (PAT). The ability of plants to perform PAT is ascribed to the auxin export activity of PIN transporters (1). Plasma membrane PINs can be restricted to a specific side of cells (2), and when this polarity is maintained in continuous plant cell files, the combined activity of identically localized PINs results in auxin flowing in that direction (3). This lays the vectorial foundations for PAT to create local auxin gradients and plant-wide PAT streams that are critical for auxin action and normal plant growth (4, 5).Synthetic PAT inhibitors such as N-1-naphthylphthalamic acid (NPA) were initially developed as herbicides and then subsequently exploited by researchers to identify and characterize the unique PAT-based mechanisms that drive plant development (6). Having been used for over six decades, the question as to how NPA actually inhibits PAT has been keenly pursued. Several putative modes of action have been proposed, but the topic remains to date not fully or satisfactorily resolved (6).Early studies established NPA binding with high affinity to membrane-integral components of plant membranes (7–10). With the later discovery of pin1 mutants bearing their distinct bare inflorescences reminiscent of NPA-treated plants (11), followed by identification of the PIN gene family and gradual confirmation that PINs were NPA-sensitive auxin transporters that mediated PAT (1–5), it was apparent that the physiological and genetic evidence overwhelmingly linked NPA to inhibition of PIN activity (6). However, direct molecular association of NPA with PINs has never been reported (6). Instead, a substantial body of data has accumulated suggesting that the NPA target is not PIN itself, but rather other proteins or complexes that either actively coparticipate in PAT or are indirectly involved in control of PAT components (6, 12). Members of the B-family of ABC transporters, such as ABCB1 and ABCB19, showed high-affinity NPA binding and NPA-sensitive auxin export (1, 12–15), thus leading to proposals that they may either physically interact with PINs, or functionally interact such that their nonpolar auxin export activity contributes to PAT and/or to regulation of PINs (12, 16). In these scenarios, PIN/PAT would be rendered vulnerable to the NPA sensitivity of ABCB. However, these schemes are not yet fully resolved, are not fully consistent with key genetic and physiological data (6), and are particularly obfuscated by ABCB1/19 functioning both interactively and independently from PINs (1, 12, 15–20), with ABCB-PIN interaction occurring in an as-yet-unclarified manner (15, 18).A further twist in assigning ABCBs as the main NPA target is their regulation by their chaperone TWD1/FKBP42 (14, 16), with TWD1 itself also being an NPA-binding protein (14, 17). NPA interferes with this regulation and affects TWD1-ABCB interaction, but curiously NPA cannot bind stably to the ABCB-TWD1 complex (14, 17). As TWD1 has also been implicated in NPA-sensitive actin-based PIN trafficking (17), this has led to a model proposing that TWD1 could mediate the NPA sensitivities of both ABCB and PINs, thus presenting TWD1 as a modulator of PAT (17, 21). In an analogous scheme in some plant species, CYPA immunophilins such as tomato DGT, which are functionally similar to TWD1/FKBP42, are suggested to replace TWD1 in modulating auxin transporters and transducing NPA effects to PINs (12, 21).Similar to TWD1, BIG/TIR3 has also been associated with NPA and PIN trafficking (22). Given the undisputed role of trafficking in controlling PIN polarity (5), these reported effects warrant attention, although they are inconsistent with other reports that NPA perturbs neither vesicular trafficking nor actin dynamics in conditions where auxin transport is inhibited (23, 24). Together with trafficking, phosphorylation is another key modulator of PIN polarity as well as activity (5), so it is not surprising to find hypotheses suggesting that NPA could interfere with critical phosphorylation events (6), particularly as PID, a kinase crucial for PIN trafficking and activation, has also been connected to ABCB function and TWD1/ABCB/NPA interactions (25). Others propose that NPA may mimic natural compounds in their capacity as endogenous regulators of PAT, with plant flavonoids being suspected candidates (6, 26). Since flavonoids can compete with or inhibit ATP-binding in mammalian kinases and ABC transporters (27, 28), and as flavonoids can bind to and inhibit PID (25), a phosphorylation-based NPA mode of action would overlap with this hypothesis and poses the question whether NPA acts similarly as an ATP mimic.With these many potential NPA-affected pathways, there is a need to distinguish between low- and high-affinity NPA targets and possible secondary effects due to prolonged PAT inhibition. Current consensus is that low concentrations of NPA (<10 µM) cause direct inhibition of auxin transporters in PAT (21) and the consequent physiological effects seen in planta (IC₅₀ 0.1 to 10 µM) (7, 9, 19, 23, 29). This is associated with high-affinity binding to membranes (K_d 0.01 to 0.1 µM) (7, 8) and the inhibition of PIN/ABCB activity in short-term auxin transport assays (1, 14, 18, 20, 23). In contrast, NPA is thought to affect trafficking (21, 30) and other non-PAT processes (31) when used at higher doses (50 to 200 µM NPA), presumably via binding to its lower-affinity targets, although excessive NPA exposure may also have fast-acting toxic side effects (23). As the in vitro affinity of TWD1 for NPA is surprisingly low (K_d ∼100 µM) (17), the TWD1-mediated NPA effects on PIN/PAT are thought to be of the low-affinity type and linked to trafficking perturbations (17, 21). However, as NPA is always externally applied to plants or cells, it is not clear how or where the drug distributes or accumulates, and thus there may be discrepancies between actual and reported/apparent effective concentrations, as might be the case for TWD1 (17). Finally, NPA also binds with low affinity to inhibit APM1, an aminopeptidase implicated in auxin-related plant growth, but as with trafficking effects, this low-affinity NPA interaction is not connected to direct regulation of PAT (31).Thus, the available data proffer various indirect mechanisms that could lead to NPA inhibition of PIN-mediated PAT, but the proposed schemes have complicating aspects and struggle at times to satisfactorily explain the prime effects of NPA. Here we propose an alternative simpler scenario involving a more direct link between NPA and PINs that would resolve some of these currently outstanding issues. We present evidence from heterologous transport assays, classical in situ membrane binding, and oligomerization studies which collectively suggest that NPA can interact directly in a high-affinity manner with PINs, leading to conformational or structural effects and inhibition of auxin export activity.     

First-in-class humanized FSH blocking antibody targets bone and fat

Blocking the action of FSH genetically or pharmacologically in mice reduces body fat, lowers serum cholesterol, and increases bone mass, making an anti-FSH agent a potential therapeutic for three global epidemics: obesity, osteoporosis, and hypercholesterolemia. Here, we report the generation, structure, and function of a first-in-class, fully humanized, epitope-specific FSH blocking antibody with a K_D of 7 nM. Protein thermal shift, molecular dynamics, and fine mapping of the FSH–FSH receptor interface confirm stable binding of the Fab domain to two of five receptor-interacting residues of the FSHβ subunit, which is sufficient to block its interaction with the FSH receptor. In doing so, the humanized antibody profoundly inhibited FSH action in cell-based assays, a prelude to further preclinical and clinical testing.

Obesity and osteoporosis affect nearly 650 million and 200 million people worldwide, respectively (1, 2). Yet the armamentarium for preventing and treating these disorders remains limited, particularly when compared with public health epidemics of a similar magnitude. It has also become increasingly clear that obesity and osteoporosis track together clinically. First, body mass does not protect against bone loss; instead, obesity can be permissive to osteoporosis and a high fracture risk (3, 4). Furthermore, the menopausal transition marks the onset not only of rapid bone loss, but also of visceral obesity and dysregulated energy balance (5–9). These physiologic aberrations have been attributed traditionally to a decline in serum estrogen, although, during the perimenopause—2 to 3 y prior to the last menstrual period—serum estrogen is within the normal range, while FSH levels rise to compensate for reduced ovarian reserve (10–12). In our view, therefore, the early skeletal and metabolic derangements cannot conceivably be explained solely by declining estrogen (13, 14).The past decade has shown that pituitary hormones can act directly on the skeleton and other tissues, a paradigm shift that is in stark contrast to previously held views on their sole regulation of endocrine targets (15–25). We and others have shown that FSH can bypass the ovary to act on G_i-coupled FSH receptors (FSHRs) on osteoclasts to stimulate bone resorption and inhibit bone formation (26, 27). This mechanism, which could underscore the bone loss during early menopause, is testified by the strong correlations between serum FSH, bone turnover, and bone mineral density (7–9, 14, 16, 26). Likewise, activating polymorphisms in the FSHR in postmenopausal women are linked to a high bone turnover and reduced bone mass (27). It therefore made biological and clinical sense to inhibit FSH action during this period to prevent bone loss.Toward this goal, we generated murine polyclonal and monoclonal antibodies to a 13-amino-acid–long binding epitope of FSHβ (28–31). The mouse and human FSHβ epitopes differ by just two amino acids; hence, blocking antibodies to the human epitope showed efficacy in mice (28). The antibodies displayed two sets of actions: they attenuated the loss of bone after ovariectomy by inhibiting bone resorption and stimulating bone formation and displayed profound effects on body composition and energy metabolism (28, 29, 31). Most notably, in a series of contemporaneously reproduced experiments, we (M.Z. and C.J.R.) found that FSH blockade reduced body fat, triggered adipocyte beiging, and increased thermogenesis in models of obesity, notably post ovariectomy and after high-fat diet (29). Our findings have been further confirmed independently by two groups who used a FSHβ–GST fusion protein or tandem repeats of the 13-amino-acid–long FSHβ epitope for studies on bone and fat, respectively (32, 33). Consistent with the mouse data, inhibiting FSH secretion using a GnRH agonist in prostate cancer patients resulted in low body fat compared with orchiectomy, wherein FSH levels are high (34). This interventional clinical trial provides evidence for a therapeutic benefit of reducing FSH levels on body fat in people. There is also new evidence that FSH blockade lowers serum cholesterol (35, 36).Thus, both emerging and validated datasets on the antiobesity, osteoprotective, and lipid-lowering actions of FSH blockade in mice and in humans prompted our current attempt to develop and characterize an array of fully humanized FSH-blocking antibodies for future testing in people. Here, we report that our lead first-in-class humanized antibody, Hu6, and two related molecules, Hu26 and Hu28, bind human FSH with a high affinity (K_Ds <10 nM), block the binding of FSH on the human FSHR, and inhibit FSH action in functional cell-based assays.     

Cognitive aging is associated with redistribution of synaptic weights in the hippocampus

Behaviors that rely on the hippocampus are particularly susceptible to chronological aging, with many aged animals (including humans) maintaining cognition at a young adult-like level, but many others the same age showing marked impairments. It is unclear whether the ability to maintain cognition over time is attributable to brain maintenance, sufficient cognitive reserve, compensatory changes in network function, or some combination thereof. While network dysfunction within the hippocampal circuit of aged, learning-impaired animals is well-documented, its neurobiological substrates remain elusive. Here we show that the synaptic architecture of hippocampal regions CA1 and CA3 is maintained in a young adult-like state in aged rats that performed comparably to their young adult counterparts in both trace eyeblink conditioning and Morris water maze learning. In contrast, among learning-impaired, but equally aged rats, we found that a redistribution of synaptic weights amplifies the influence of autoassociational connections among CA3 pyramidal neurons, yet reduces the synaptic input onto these same neurons from the dentate gyrus. Notably, synapses within hippocampal region CA1 showed no group differences regardless of cognitive ability. Taking the data together, we find the imbalanced synaptic weights within hippocampal CA3 provide a substrate that can explain the abnormal firing characteristics of both CA3 and CA1 pyramidal neurons in aged, learning-impaired rats. Furthermore, our work provides some clarity with regard to how some animals cognitively age successfully, while others’ lifespans outlast their “mindspans.”

Aging is the biggest risk factor for Alzheimer’s disease, but many aged individuals nevertheless retain the ability to perform cognitive tasks with young adult (YA)-like competency, and are thus resilient to age-related cognitive decline and dementias (1, 2). The mechanisms of such resilience are unknown, but are thought to involve neural or cognitive reserve, brain network adaptations, or simply the ability to maintain cognitive brain circuits in a YA-like state (3–5). Much of the cellular and functional insight into the concept or risk of/resilience against age-related cognitive impairments has come from animal models of normal/nonpathological aging (6–10). Many of these studies have shown that circuit function abnormalities are associated with behavioral impairments. The cellular and structural bases for such functional aberrations, however, remain largely unknown.Two of the most well-studied cognitive domains that show susceptibility to chronological aging in both rodents and nonhuman primates are working memory and spatial/temporal memory (6–10). Importantly, these cognitive domains engage anatomically distinct neurocognitive systems, with the former relying on prefrontal/orbitofrontal cortical circuits and the latter relying on hippocampal circuitry. Interestingly, although behavioral deficits in these two domains (in the case of rat models of cognitive aging) begin to emerge, worsen, and become increasingly prevalent between 12 and 18 mo of age in most strains (reviewed in refs. 9 and 11), cognitive aging within hippocampus-dependent forms of learning and memory are relatively independent of those that engage the prefrontal/orbitofrontal cortical neural systems (6–9, 12–15).Neither the mechanisms underlying the conservation of memory function across chronological aging nor those contributing to the age-related emergence and exacerbation of memory impairments are clearly understood for either neurocognitive system. It is clear, however, that neither frank neuronal loss (16, 17) nor overall synapse loss (18) contributes to cognitive aging within the medial temporal lobe/hippocampal memory system. Rather, the intriguing idea that has emerged from work in both the hippocampal and the prefrontal/orbitofrontal cortical memory systems is that there are functional alterations in the synaptic connections in individual microcircuits embedded within these larger neuroanatomical systems (6–10, 19–31).Axospinous synapses (including those in hippocampal and cortical circuits) are characterized on the basis of the three-dimensional morphology of their postsynaptic densities (PSDs) (20, 32–34). The most-abundant axospinous synaptic subtype has a continuous, macular, disk-shaped PSD, as compared to the less-abundant perforated synaptic subtype, which has at least one discontinuity in its PSD (34). In addition to differing substantially with regard to relative frequency, perforated and nonperforated synapses also harbor major differences in size and synaptic AMPA-type and NMDA-type receptor expression levels (AMPAR and NMDAR, respectively) (34–38). There is also evidence that perforated and nonperforated synapses are differentially involved in synaptic plasticity (39–44) and in preservation of—or reductions in—memory function during chronological aging (6, 20, 45). Layered onto these general distinctions between perforated and nonperforated synapses are more specific differences in their characteristics when considered within neural circuits. For example, perforated synapses have a stronger and more consistent influence on neuronal computation within hippocampal region CA1 than their nonperforated counterparts, which nevertheless outnumber the former by a roughly 9-to-1 ratio (34, 46, 47).These and other circuit-specific differences necessitate a circuit-based approach to understanding the synaptic bases underlying the retention or loss of YA-like memory function in the aging brain. In many ways, the hippocampal system is particularly convenient for such circuit-based approaches (48, 49). Information about the internal and external world is funneled to the parahippocampal system and then relayed via the entorhinal cortex to the dentate gyrus, the first component of the so-called trisynaptic circuit in the hippocampus proper. Granule cells in the dentate gyrus then transmit their computations to hippocampal region CA3 via the mossy fibers, which form very large and anatomically distinct synapses called mossy fiber bouton–thorny excrescence synaptic complexes in the stratum lucidum (SL). CA3 pyramidal neurons then integrate information from their autoassociational connections in the stratum radiatum (SR) and stratum oriens (SO), with both direct entorhinal inputs in stratum lacunosum-moleculare (SLM) and the dentate gyrus inputs in the SL, and convey this information to hippocampal CA1 pyramidal neurons. Neurons in hippocampal CA1 then integrate this information in their basal and apical SR dendrites with direct entorhinal cortical inputs in their most distal, tufted dendrites in the SLM, and represent the first and largest extrahippocampal output from the hippocampus proper. Thus, the computations performed both within individual hippocampal subregions and between them as an interconnected neurocognitive system are complex, and involve a combination of intrinsic (i.e., membrane-bound ion channels that regulate membrane excitability) and synaptic (i.e., ligand-gated ion channels expressed at both excitatory and inhibitory synapses) influences. Additionally, age-related changes at any level of these complex circuits will have downstream consequences on the accuracy/reliability of the information being relayed to extrahippocampal regions via CA1 pyramidal neurons.Given the amount of evidence supporting a possible synaptic explanation for age-related learning and memory impairments in hippocampus-dependent forms of cognition (6–10), we combined patch-clamp physiology, serial section conventional and immunogold electron microscopy (EM), quantitative Western blot analyses, and behavioral characterization using two hippocampus-dependent forms of learning in YA (6- to 8-mo old) and aged rats (28- to 29-mo old) to examine two interconnected hippocampal regions implicated in cognitive aging: Regions CA1 and CA3. We focused on CA1 and CA3 because of their central location in the hippocampal circuit (48–50), their similar laminar dendritic structure (48–50), and their well-documented age-related changes in place field specificity and reliability (51–56).We find that the synaptic architecture and balance of synaptic weights in YA and aged, learning-unimpaired (AU) rats is remarkably similar, but that both are different in aged, learning-impaired (AI) rats. Moreover, this restructuring among “unsuccessful” cognitive agers has an intriguing specificity: It involves only AMPARs, only perforated axospinous synapses, and only hippocampal region CA3, which together shift the balance of synaptic weights that drive action potential output in CA3 pyramidal neurons maladaptively toward an overemphasis of the autoassociational synapses that interconnect CA3 pyramidal neurons.     

Cholinergic suppression of hippocampal sharp-wave ripples impairs working memory

Learning and memory are assumed to be supported by mechanisms that involve cholinergic transmission and hippocampal theta. Using G protein–coupled receptor-activation–based acetylcholine sensor (GRAB_ACh3.0) with a fiber-photometric fluorescence readout in mice, we found that cholinergic signaling in the hippocampus increased in parallel with theta/gamma power during walking and REM sleep, while ACh3.0 signal reached a minimum during hippocampal sharp-wave ripples (SPW-R). Unexpectedly, memory performance was impaired in a hippocampus-dependent spontaneous alternation task by selective optogenetic stimulation of medial septal cholinergic neurons when the stimulation was applied in the delay area but not in the central (choice) arm of the maze. Parallel with the decreased performance, optogenetic stimulation decreased the incidence of SPW-Rs. These findings suggest that septo–hippocampal interactions play a task-phase–dependent dual role in the maintenance of memory performance, including not only theta mechanisms but also SPW-Rs.

The neurotransmitter acetylcholine is thought to be critical for hippocampus-dependent declarative memories (1, 2). Reduction in cholinergic neurotransmission, either in Alzheimer’s disease or in experiments with cholinergic antagonists, such as scopolamine, impairs memory function (3–8). Acetylcholine may bring about its beneficial effects on memory encoding by enhancing theta rhythm oscillations, decreasing recurrent excitation, and increasing synaptic plasticity (9–11). Conversely, drugs which activate cholinergic receptors enhance learning and, therefore, are a neuropharmacological target for the treatment of memory deficits in Alzheimer’s disease (5, 12, 13).The contribution of cholinergic mechanisms in the acquisition of long-term memories and the role of the hippocampal–entorhinal–cortical interactions are well supported by experimental data (5, 12, 13). In addition, working memory or “short-term” memory is also supported by the hippocampal–entorhinal–prefrontal cortex (14–16). Working memory in humans is postulated to be a conscious process to “keep things in mind” transiently (16). In rodents, matching to sample task, spontaneous alternation between reward locations, and the radial maze task have been suggested to function as a homolog of working memory [“working memory like” (17)].Cholinergic activity is a critical requirement for working memory (18, 19) and for sustaining theta oscillations (10, 20–22). In support of this contention, theta–gamma coupling and gamma power are significantly higher in the choice arm of the maze, compared with those in the side arms where working memory is no longer needed for correct performance (23–26). It has long been hypothesized that working memory is maintained by persistent firing of neurons, which keep the presented items in a transient store in the prefrontal cortex and hippocampal–entorhinal system (27–31), although the exact mechanisms are debated (32–37). An alternative hypothesis holds that items of working memory are stored in theta-nested gamma cycles (38). Common in these models of working memory is the need for an active, cholinergic system–dependent mechanism (39–41). However, in spontaneous alternation tasks, the animals are not moving continuously during the delay, and theta oscillations are not sustained either. During the immobility epochs, theta is replaced by intermittent sharp-wave ripples (SPW-R), yet memory performance does not deteriorate. On the contrary, artificial blockade of SPW-Rs can impair memory performance (42, 43), and prolongation of SPW-Rs improves performance (44). Under the cholinergic hypothesis of working memory, such a result is unexpected.To address the relationship between cholinergic/theta versus SPW-R mechanism in spontaneous alternation, we used a G protein–coupled receptor-activation–based acetylcholine sensor (GRAB_ACh3.0) (45) to monitor acetylcholine (ACh) activity during memory performance in mice. In addition, we optogenetically enhanced cholinergic tone, which suppresses SPW-Rs by a different mechanism than electrically or optogenetically induced silencing of neurons in the hippocampus (43, 44). We show that cholinergic signaling in the hippocampus increases in parallel with theta power/score during walking and rapid eye movement (REM) sleep and reaches a transient minimum during SPW-Rs. Selective optogenetic stimulation of medial septal cholinergic neurons decreased the incidence of SPW-Rs during non-REM sleep (46–48), as well as during the delay epoch of a working memory task and impaired memory performance. These findings demonstrate that memory performance is supported by complementary theta and SPW-R mechanisms.     

Adipose tissue is a critical regulator of osteoarthritis

Osteoarthritis (OA), the leading cause of pain and disability worldwide, disproportionally affects individuals with obesity. The mechanisms by which obesity leads to the onset and progression of OA are unclear due to the complex interactions among the metabolic, biomechanical, and inflammatory factors that accompany increased adiposity. We used a murine preclinical model of lipodystrophy (LD) to examine the direct contribution of adipose tissue to OA. Knee joints of LD mice were protected from spontaneous or posttraumatic OA, on either a chow or high-fat diet, despite similar body weight and the presence of systemic inflammation. These findings indicate that adipose tissue itself plays a critical role in the pathophysiology of OA. Susceptibility to posttraumatic OA was reintroduced into LD mice using implantation of a small adipose tissue depot derived from wild-type animals or mouse embryonic fibroblasts that undergo spontaneous adipogenesis, implicating paracrine signaling from fat, rather than body weight, as a mediator of joint degeneration.

Osteoarthritis (OA) is the leading cause of pain and disability worldwide and is associated with increased all-cause mortality and cardiovascular disease (1, 2). OA is strongly associated with obesity, suggesting that either increased biomechanical joint loading or systemic inflammation and metabolic dysfunction related to obesity are responsible for joint degeneration (1, 2). However, increasing evidence is mounting that changes in biomechanical loading due to increased body mass do not account for the severity of obesity-induced knee OA (1–9). These observations suggest that other factors related to the presence of adipose tissue and adipose tissue-derived cytokines—termed adipokines—play critical roles in this process and other musculoskeletal conditions (1, 2, 6, 7, 10). As there are presently no disease-modifying OA drugs available, direct evidence linking adipose tissue and cartilage health could provide important mechanistic insight into the natural history of OA and obesity and therefore guide the development and translation of novel OA therapeutic strategies designed to preserve joint health.The exact contribution of the adipokine-signaling network in OA has been difficult to determine due to the complex interactions among metabolic, biomechanical, and inflammatory factors related to obesity (11). To date, the link between increased adipose tissue mass and OA pathogenesis has largely been correlative (6, 7, 12), and, as such, the direct effect of adipose tissue and the adipokines it releases has been difficult to separate from other factors such as dietary composition or excess body mass in the context of obesity, which is most commonly caused by excessive nutrition (2, 6, 7). In particular, leptin, a proinflammatory adipokine and satiety hormone secreted proportionally to adipose tissue mass is most consistently increased in obesity-induced OA (1), and leptin knockout mice are protected from OA (6, 7). However, it remains to be determined whether leptin directly contributes to OA pathogenesis, independent of its effect on metabolism (and weight). Additional adipokines that have been implicated in the onset and progression of OA include adiponectin, resistin, visfatin, chimerin, and inflammatory cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α) (13). The infrapatellar fat pad represents a local source of adipokines within the knee joint, but several studies indicate strong correlations with visceral adipose tissue, outside of the joint organ system, with OA severity (14). Furthermore, adipokine receptors are found on almost all cells within the joint and, therefore, could directly contribute to OA pathogenesis through synovitis, cartilage damage, and bone remodeling (13). The role of other adipokines (15) in OA pathogenesis remains to be determined, as it has been difficult to separate and directly test the role of adipokines from other biomechanical, inflammatory, and metabolic factors that contribute to OA pathogenesis.To directly investigate the mechanisms by which adipose tissue affects OA, we used a transgenic mouse with lipodystrophy (LD) that completely lacks adipose tissue and, therefore, adipokine signaling. The LD model system affords the unique opportunity to directly examine the effects of adipose tissue and its secretory factors on musculoskeletal pathology without the confounding effect of diet (16, 17). While LD mice completely lack adipose tissue depots, they demonstrate similar body mass to wild-type (WT) controls on a chow diet (12, 16–19). These characteristics provide a unique model that can be used to eliminate the factor of loading due to body mass on joint damage and, thus, to directly test the effects of fat and factors secreted by fat on musculoskeletal tissues. Of particular interest, LD mice also exhibit several characteristics that have been associated with OA, including sclerotic bone (11, 20), metabolic derangement (3, 5, 7–9, 21, 22), and muscle weakness (2). Despite these OA-predisposing features, LD mice are protected from OA and implantation of adipose tissue back into LD mice restores susceptibility to OA—demonstrating a direct relationship between adipose tissue and cartilage health, independent of the effect of obesity on mechanical joint loading.     

Tropospheric carbonyl sulfide mass balance based on direct measurements of sulfur isotopes

Robust estimates for the rates and trends in terrestrial gross primary production (GPP; plant CO₂ uptake) are needed. Carbonyl sulfide (COS) is the major long-lived sulfur-bearing gas in the atmosphere and a promising proxy for GPP. Large uncertainties in estimating the relative magnitude of the COS sources and sinks limit this approach. Sulfur isotope measurements (³⁴S/³²S; δ³⁴S) have been suggested as a useful tool to constrain COS sources. Yet such measurements are currently scarce for the atmosphere and absent for the marine source and the plant sink, which are two main fluxes. Here we present sulfur isotopes measurements of marine and atmospheric COS, and of plant-uptake fractionation experiments. These measurements resulted in a complete data-based tropospheric COS isotopic mass balance, which allows improved partition of the sources. We found an isotopic (δ³⁴S ± SE) value of 13.9 ± 0.1‰ for the troposphere, with an isotopic seasonal cycle driven by plant uptake. This seasonality agrees with a fractionation of −1.9 ± 0.3‰ which we measured in plant-chamber experiments. Air samples with strong anthropogenic influence indicated an anthropogenic COS isotopic value of 8 ± 1‰. Samples of seawater-equilibrated-air indicate that the marine COS source has an isotopic value of 14.7 ± 1‰. Using our data-based mass balance, we constrained the relative contribution of the two main tropospheric COS sources resulting in 40 ± 17% for the anthropogenic source and 60 ± 20% for the oceanic source. This constraint is important for a better understanding of the global COS budget and its improved use for GPP determination.

The Earth system is going through rapid changes as the climate warms and CO₂ level rises. This rise in CO₂ is mitigated by plant uptake; hence, it is important to estimate global and regional photosynthesis rates and trends (1). Yet, robust tools for investigating these processes at a large scale are scarce (2). Recent studies suggest that carbonyl sulfide (COS) could provide an improved constraint on terrestrial photosynthesis (gross primary production, GPP) (2–12). COS is the major long-lived sulfur-bearing gas in the atmosphere and the main supplier of sulfur to the stratospheric sulfate aerosol layer (13), which exerts a cooling effect on the Earth’s surface and regulates stratospheric ozone chemistry (14).During terrestrial photosynthesis, COS diffuses into leaf stomata and is consumed by photosynthetic enzymes in a similar manner to CO₂ (3–5). Contrary to CO₂, COS undergoes rapid and irreversible hydrolysis mainly by the enzyme carbonic-anhydrase (6, 7). Thus, COS can be used as a proxy for the one-way flux of CO₂ removal from the atmosphere by terrestrial photosynthesis (2, 8–11). However, the large uncertainties in estimating the COS sources weaken this approach (10–12, 15). Tropospheric COS has two main sources: the oceans and anthropogenic emissions, and one main sink–terrestrial plant uptake (8, 10–13). Smaller sources include biomass burning, soil emissions, wetlands, volcanoes, and smaller sinks include OH destruction, stratospheric destruction, and soil uptake (12). The largest source of COS to the atmosphere is the ocean, both as direct COS emission, and as indirect carbon disulfide (CS₂) and dimethylsulfide (DMS) emissions that are rapidly oxidized to COS (10, 16–20). Recent studies suggest oceanic COS emissions are in the range of 200–4,000 GgS/y (19–22). The second major COS source is the anthropogenic source, which is dominated by indirect emissions derived from CS₂ oxidation, mainly from the use of CS₂ as an industrial solvent. Direct emissions of COS are mainly derived from coal and fuel combustion (17, 23, 24). Recent studies suggest that anthropogenic emissions are in the range of 150–585 GgS/y (23, 24). The terrestrial plant uptake is estimated to be in the range of 400–1,360 GgS/y (11). Measurements of sulfur isotope ratios (δ³⁴S) in COS may be used to track COS sources and thus reduce the uncertainties in their flux estimations (15, 25–27). However, the isotopic mass balance approach works best if the COS end members are directly measured and have a significantly different isotopic signature. Previous δ³⁴S measurements of atmospheric COS are scarce and there have been no direct measurements of two important components: the δ³⁴S of oceanic COS emissions, and the isotopic fractionation of COS during plant uptake (15, 25–27). In contrast to previous studies that used assessments for these isotopic values, our aim was to directly measure the isotopic values of these missing components, and to determine the tropospheric COS δ³⁴S variability over space and time.