Natural cystatin C fragments inhibit GPR15-mediated HIV and SIV infection without interfering with GPR15L signaling

GPR15 is a G protein-coupled receptor (GPCR) proposed to play a role in mucosal immunity that also serves as a major entry cofactor for HIV-2 and simian immunodeficiency virus (SIV). To discover novel endogenous GPR15 ligands, we screened a hemofiltrate (HF)-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2, and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4⁺ T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this GPCR.

G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins involved in the transduction of signals from the extracellular environment into the cell and play key roles in immune responses, homeostasis, metabolism, and organogenesis (1, 2). Besides their physiological roles, some GPCRs also represent important coreceptors for HIV and/or simian immunodeficiency virus (SIV) entry. HIV-1, the main causative agent of AIDS, utilizes C-C chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) as major entry cofactors (3–8). The chemokine ligands CCL5 (also known as RANTES) and CXCL12 (also named SDF-1) inhibit CCR5- or CXCR4-mediated HIV-1 infection, respectively. Thus, we have previously taken advantage of HIV-1 entry to examine complex blood-derived peptide libraries for novel naturally occurring ligands of CCR5 and CXCR4 (9, 10). Initially, we identified a truncated form of the chemokine C-C ligand 14 (CCL14) as a CCR5 agonist and potent inhibitor of CCR5-tropic HIV-1 strains (11, 12). More recently, we discovered a small fragment of human serum albumin (EPI-X4) as an effective and highly specific CXCR4 antagonist and inhibitor of CXCR4-tropic HIV-1 strains (13). While HIV-1 coreceptor utilization is mainly restricted to CCR5 and CXCR4, HIV-2 and SIVs are more promiscuous in their entry cofactor usage. For example, many HIV-2 and SIV strains utilize BOB/GPR15 and Bonzo/STRL-33/CXCR6 in addition to CCR5 and/or CXCR4 for viral entry into CD4⁺ target cells (14–20). GPR15 is a GPCR reported to regulate T cell trafficking to the colon that may play a role in intestinal homeostasis and inflammation (21, 22). Recently, an agonistic C-C chemokine ligand of GPR15, named GPR15L, has been characterized (23, 24). GPR15L is expressed in colon and cervical epithelia and might play a role in mucosal immunity.To discover novel endogenous GPR15 ligands, we screened a hemofiltrate (HF)-derived peptide library containing essentially all peptides and small proteins circulating in human blood in their final processed and physiologically relevant forms (10) for inhibitors of GPR15-mediated SIV infection. Multiple rounds of peptide separation and antiviral screening identified a C-terminal fragment of cystatin C (named CysC95-146) as a potent and specific inhibitor of GPR15-dependent HIV and SIV infection. Cystatin C is a small (13 kDa) basic protein that is produced by all nucleated cells (25) and represents the most abundant and potent extracellular inhibitor of cysteine proteases (26). It is found in virtually all tissues and body fluids and commonly used as a marker of renal function (27). Accumulating evidence suggests a role of cystatin C in inflammation, neutrophil chemotaxis, and resistance to bacterial as well as viral infections (28–31). We show that cystatin C fragments preventing GPR15-dependent HIV and SIV infection are generated by proteases activated during antiviral immune responses. Unexpectedly, the recently discovered chemokine ligand of GPR15, GPR15L (23, 24), had no significant effect on viral entry. In addition, CysC95-146 prevented SIV and HIV-2 infection without interfering with GPR15L-mediated signaling. Our data support that naturally occurring cystatin C fragments are capable of blocking GPR15-mediated primate lentiviral infection without interfering with the physiological signaling function of this GPCR.     

From the Cover: Evidence from South Africa for a protracted end-Permian extinction on land

Earth’s largest biotic crisis occurred during the Permo–Triassic Transition (PTT). On land, this event witnessed a turnover from synapsid- to archosauromorph-dominated assemblages and a restructuring of terrestrial ecosystems. However, understanding extinction patterns has been limited by a lack of high-precision fossil occurrence data to resolve events on submillion-year timescales. We analyzed a unique database of 588 fossil tetrapod specimens from South Africa’s Karoo Basin, spanning ∼4 My, and 13 stratigraphic bin intervals averaging 300,000 y each. Using sample-standardized methods, we characterized faunal assemblage dynamics during the PTT. High regional extinction rates occurred through a protracted interval of ∼1 Ma, initially co-occurring with low origination rates. This resulted in declining diversity up to the acme of extinction near the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary. Regional origination rates increased abruptly above this boundary, co-occurring with high extinction rates to drive rapid turnover and an assemblage of short-lived species symptomatic of ecosystem instability. The “disaster taxon” Lystrosaurus shows a long-term trend of increasing abundance initiated in the latest Permian. Lystrosaurus comprised 54% of all specimens by the onset of mass extinction and 70% in the extinction aftermath. This early Lystrosaurus abundance suggests its expansion was facilitated by environmental changes rather than by ecological opportunity following the extinctions of other species as commonly assumed for disaster taxa. Our findings conservatively place the Karoo extinction interval closer in time, but not coeval with, the more rapid marine event and reveal key differences between the PTT extinctions on land and in the oceans.

Mass extinctions are major perturbations of the biosphere resulting from a wide range of different causes including glaciations and sea level fall (1), large igneous provinces (2), and bolide impacts (3, 4). These events caused permanent changes to Earth’s ecosystems, altering the evolutionary trajectory of life (5). However, links between the broad causal factors of mass extinctions and the biological and ecological disturbances that lead to species extinctions have been difficult to characterize. This is because ecological disturbances unfold on timescales much shorter than the typical resolution of paleontological studies (6), particularly in the terrestrial record (6–8). Coarse-resolution studies have demonstrated key mass extinction phenomena including high extinction rates and lineage turnover (7, 9), changes in species richness (10), ecosystem instability (11), and the occurrence of disaster taxa (12). However, finer time resolutions are central to determining the association and relative timings of these effects, their potential causal factors, and their interrelationships. Achieving these goals represents a key advance in understanding the ecological mechanisms of mass extinctions.The end-Permian mass extinction (ca. 251.9 Ma) was Earth’s largest biotic crisis as measured by taxon last occurrences (13–15). Large outpourings from Siberian Trap volcanism (2) are the likely trigger of calamitous climatic changes, including a runaway greenhouse effect and ocean acidification, which had profound consequences for life on land and in the oceans (16–18). An estimated 81% of marine species (19) and 89% of tetrapod genera became extinct as established Permian ecosystems gave way to those of the Triassic. In the ocean, this included the complete extinction of reef-forming tabulate and rugose corals (20, 21) and significant losses in previously diverse ammonoid, brachiopod, and crinoid families (22). On land, many nonmammalian synapsids became extinct (16), and the glossopterid-dominated floras of Gondwana also disappeared (23). Stratigraphic sequences document a global “coral gap” and “coal gap” (24, 25), suggesting reef and forest ecosystems were rare or absent for up to 5 My after the event (26). Continuous fossil-bearing deposits documenting patterns of turnover across the Permian–Triassic transition (PTT) on land (27) and in the oceans (28) are geographically widespread (29, 30), including marine and continental successions that are known from China (31, 32) and India (33). Continental successions are known from Russia (34), Australia (35), Antarctica (36), and South Africa’s Karoo Basin (Fig. 1 and 37–40), the latter providing arguably the most densely sampled and taxonomically scrutinized (41–43) continental record of the PTT. The main extinction has been proposed to occur at the boundary between two biostratigraphic zones with distinctive faunal assemblages, the Daptocephalus and Lystrosaurus declivis assemblage zones (Fig. 1), which marks the traditional placement of the Permian–Triassic geologic boundary [(37) but see ref. 44]. Considerable research has attempted to understand the anatomy of the PTT in South Africa (38, 39, 45–52) and to place it in the context of biodiversity changes across southern Gondwana (53, 54) and globally (29, 31, 32, 44, 47, 55).Open in a separate windowFig. 1.Map of South Africa depicting the distribution of the four tetrapod fossil assemblage zones (Cistecephalus, Daptocephalus, Lystrosaurus declivis, Cynognathus) and our two study sites where fossils were collected in this study (sites A and B). Regional lithostratigraphy and biostratigraphy within the study interval are shown alongside isotope dilution–thermal ionization mass spectrometry dates retrieved by Rubidge et al., Botha et al., and Gastaldo et al. (37, 44, 80). The traditional (dashed red line) and associated PTB hypotheses for the Karoo Basin (37, 44) are also shown. Although traditionally associated with the PTB, the Daptocephalus–Lystrosaurus declivis Assemblage Zone boundary is defined by first appearances of co-occurring tetrapod assemblages, so its position relative to the three PTB hypotheses is unchanged. The Ripplemead member (*) has yet to be formalized by the South African Committee for Stratigraphy.Decades of research have demonstrated the richness of South Africa’s Karoo Basin fossil record, resulting in hundreds of stratigraphically well-documented tetrapod fossils across the PTT (37, 39, 56). This wealth of data has been used qualitatively to identify three extinction phases and an apparent early postextinction recovery phase (39, 45, 51). Furthermore, studies of Karoo community structure and function have elucidated the potential role of the extinction and subsequent recovery in breaking the incumbency of previously dominant clades, including synapsids (11, 57). Nevertheless, understanding patterns of faunal turnover and recovery during the PTT has been limited by the scarcity of quantitative investigations. Previous quantitative studies used coarsely sampled data (i.e., assemblage zone scale, 2 to 3 Ma time intervals) to identify low species richness immediately after the main extinction, potentially associated with multiple “boom and bust” cycles of primary productivity based on δ¹³C variation during the first 5 My of the Triassic (41, 58). However, many details of faunal dynamics in this interval remain unknown. Here, we investigate the dynamics of this major tetrapod extinction at an unprecedented time resolution (on the order of hundreds of thousands of years), using sample-standardized methods to quantify multiple aspects of regional change across the Cistecephalus, Daptocephalus, and Lystrosaurus declivis assemblage zones.     

Forelimb force direction and magnitude independently controlled by spinal modules in the macaque

Modular organization of the spinal motor system is thought to reduce the cognitive complexity of simultaneously controlling the large number of muscles and joints in the human body. Although modular organization has been confirmed in the hindlimb control system of several animal species, it has yet to be established in the forelimb motor system or in primates. Expanding upon experiments originally performed in the frog lumbar spinal cord, we examined whether costimulation of two sites in the macaque monkey cervical spinal cord results in motor activity that is a simple linear sum of the responses evoked by stimulating each site individually. Similar to previous observations in the frog and rodent hindlimb, our analysis revealed that in most cases (77% of all pairs) the directions of the force fields elicited by costimulation were highly similar to those predicted by the simple linear sum of those elicited by stimulating each site individually. A comparable simple summation of electromyography (EMG) output, especially in the proximal muscles, suggested that this linear summation of force field direction was produced by a spinal neural mechanism whereby the forelimb motor output recruited by costimulation was also summed linearly. We further found that the force field magnitudes exhibited supralinear (amplified) summation, which was also observed in the EMG output of distal forelimb muscles, implying a novel feature of primate forelimb control. Overall, our observations support the idea that complex movements in the primate forelimb control system are made possible by flexibly combined spinal motor modules.

To execute voluntary movement, the central nervous system (CNS) must convert the desired movement into signals that control muscles and thus the actual position of the body in space. Large numbers of possible movements (i.e., large degrees of freedom, DOF), nonlinear dynamics, and sensory delays in the musculoskeletal apparatus make voluntary movements a computational challenge. How the CNS accomplishes this conversion is a long-standing question in the field of motor control. A hierarchical and modular organization of the CNS for controlling movement might simplify this process. In this scheme, a limited number of output modules in the spinal cord would control different but overlapping sets of muscles and joints, thereby decreasing the number of variables directly controlled by the cerebral cortex. By recruiting different output modules simultaneously but independently, the CNS might take advantage of this modular structure and achieve the flexibility necessary to control a variety of behaviors at a lower computational cost than is accrued by controlling each muscle independently (1–3). Bizzi and colleagues (1, 4, 5) pioneered the original experimental evaluation of this hypothesis, showing that hindlimb movement generated by costimulation (simultaneous stimulation) of two sites in the frog lumbar spinal cord could be explained by a simple linear sum of the responses evoked by stimulating each site individually. Based on this seminal work, they further hypothesized that the supraspinal structure might control the large number of DOF by activating and combining different motor “modules” in the spinal cord (3, 6, 7). Subsequent work in the frog (8–10), rodent (11, 12), and cat (13) confirmed these results. In this paper, we tested whether the same scheme exists in the primate forelimb-control system.Today, the concept of modular organization is used as a basis in studies of human movement (14, 15). Indeed, a number of different human joint movements and muscle activities have been characterized based on this idea (i.e., muscle synergy) (16, 17), and it has even spread to fields of clinical research (18, 19) and robotic engineering (20–22). Although these studies implicitly assume that the concept of modular motor control derived from frogs and small mammals also applies to humans, few studies have attempted to directly determine whether such control is present in the primate spinal circuitry. We recently reported that cervical spinal interneurons in monkeys coactivate multiple finger muscles (23) with several time-varying profiles (24) and that these coactivations could correspond to muscle synergies in primate hand muscles (25). These findings seem to support the idea that primate forelimb movement is also controlled through modular organization and raise the possibility that efficient control of the primate limb is accomplished via brain activation of different sets of spinal cord modules (26, but see refs. 27 and 28), as previously seen in the legs of frogs (5) and rodents (11).To test this idea, we reproduced the original experimental paradigm in the cervical spinal cord (for control of forelimbs rather than hindlimbs) of anesthetized monkeys, measuring the direction and magnitude of the force field generated by costimulation as well as the electromyography (EMG) output (1, 4, 5). We tested the effect of electrically stimulating pairs of intraspinal sites both individually and simultaneously by comparing the evoked force fields measured at the wrist joint as well as the activity of several forelimb muscles. As in the lumbar spinal cord of other species (5, 11, 12), we found that the directionality of the force field evoked after simultaneous stimulation could be explained by linearly summing the force fields generated when each intraspinal site was stimulated separately. We further confirmed a comparable linear summation in the EMG output of proximal forelimb muscles as the potential source of the force fields’ linear summation. Additionally, we unexpectedly found supralinear facilitation of force field magnitude, which has not been reported in previous studies. We discuss a potential source and functional significance of this supralinear facilitation.     

Violet light suppresses lens-induced myopia via neuropsin (OPN5) in mice

Myopia has become a major public health concern, particularly across much of Asia. It has been shown in multiple studies that outdoor activity has a protective effect on myopia. Recent reports have shown that short-wavelength visible violet light is the component of sunlight that appears to play an important role in preventing myopia progression in mice, chicks, and humans. The mechanism underlying this effect has not been understood. Here, we show that violet light prevents lens defocus–induced myopia in mice. This violet light effect was dependent on both time of day and retinal expression of the violet light sensitive atypical opsin, neuropsin (OPN5). These findings identify Opn5-expressing retinal ganglion cells as crucial for emmetropization in mice and suggest a strategy for myopia prevention in humans.

Myopia (nearsightedness) in school-age children is generally axial myopia, which is the consequence of elongation of the eyeball along the visual axis. This shape change results in blurred vision but can also lead to severe complications including cataract, retinal detachment, myopic choroidal neovascularization, glaucoma, and even blindness (1–3). Despite the current worldwide pandemic of myopia, the mechanism of myopia onset is still not understood (4–8). One hypothesis that has earned a current consensus is the suggestion that a change in the lighting environment of modern society is the cause of myopia (9, 10). Consistent with this, outdoor activity has a protective effect on myopia development (9, 11, 12), though the main reason for this effect is still under debate (7, 12, 13). One explanation is that bright outdoor light can promote the synthesis and release of dopamine in the eye, a myopia-protective neuromodulator (14–16). Another suggestion is that the distinct wavelength composition of sunlight compared with fluorescent or LED (light-emitting diode) artificial lighting may influence myopia progression (9, 10). Animal studies have shown that different wavelengths of light can affect the development of myopia independent of intensity (17, 18). The effects appear to be distinct in different species: for chicks and guinea pigs, blue light showed a protective effect on experimentally induced myopia, while red light had the opposite effect (18–22). For tree shrews and rhesus monkeys, red light is protective, and blue light causes dysregulation of eye growth (23–25).It has been shown that visible violet light (VL) has a protective effect on myopia development in mice, in chick, and in human (10, 26, 27). According to Commission Internationale de l’Eclairage (International Commission on Illumination), VL has the shortest wavelength of visible light (360 to 400 nm). These wavelengths are abundant in outside sunlight but can only rarely be detected inside buildings. This is because the ultraviolet (UV)-protective coating on windows blocks all light below 400 nm and because almost no VL is emitted by artificial light sources (10). Thus, we hypothesized that the lack of VL in modern society is one reason for the myopia boom (9, 10, 26).In this study, we combine a newly developed lens-induced myopia (LIM) model with genetic manipulations to investigate myopia pathways in mice (28, 29). Our data confirm (10, 26) that visible VL is protective but further show that delivery of VL only in the evening is sufficient for the protective effect. In addition, we show that the protective effect of VL on myopia induction requires OPN5 (neuropsin) within the retina. The absence of retinal Opn5 prevents lens-induced, VL-dependent thickening of the choroid, a response thought to play a key role in adjusting the size of the eyeball in both human and animal myopia models (30–33). This report thus identifies a cell type, the Opn5 retinal ganglion cell (RGC), as playing a key role in emmetropization. The requirement for OPN5 also explains why VL has a protective effect on myopia development.     

Primate phageomes are structured by superhost phylogeny and environment

Humans harbor diverse communities of microorganisms, the majority of which are bacteria in the gastrointestinal tract. These gut bacterial communities in turn host diverse bacteriophage (hereafter phage) communities that have a major impact on their structure, function, and, ultimately, human health. However, the evolutionary and ecological origins of these human-associated phage communities are poorly understood. To address this question, we examined fecal phageomes of 23 wild nonhuman primate taxa, including multiple representatives of all the major primate radiations. We find relatives of the majority of human-associated phages in wild primates. Primate taxa have distinct phageome compositions that exhibit a clear phylosymbiotic signal, and phage–superhost codivergence is often detected for individual phages. Within species, neighboring social groups harbor compositionally and evolutionarily distinct phageomes, which are structured by superhost social behavior. Captive nonhuman primate phageome composition is intermediate between that of their wild counterparts and humans. Phage phylogenies reveal replacement of wild great ape–associated phages with human-associated ones in captivity and, surprisingly, show no signal for the persistence of wild-associated phages in captivity. Together, our results suggest that potentially labile primate-phage associations have persisted across millions of years of evolution. Across primates, these phylosymbiotic and sometimes codiverging phage communities are shaped by transmission between groupmates through grooming and are dramatically modified when primates are moved into captivity.

Mammals harbor diverse communities of microorganisms, the majority of which are bacteria in the gastrointestinal tract. Gut bacterial communities in turn host diverse phage communities that influence their structure, function, colonization patterns, and ultimately superhost health [the superhost is the host for bacteria that in turn host the phages (1)]. For example, enriched phage communities in human intestinal mucus can act as an acquired immune system by limiting mucosal bacterial populations (2), while dysbiotic gut phageomes are associated with health conditions such as type II diabetes (3), colitis (4), and stunting (5). Transplantation of healthy viral filtrates restored health in Clostridioides difficile patients (6), while in vitro studies suggest phages from stunted children shape bacterial populations differently from those of healthy children (5), supporting a direct link between phageome composition and disease. However, despite their importance in gut microbial ecosystems, the ecological and evolutionary processes that gave rise to these communities remain poorly resolved. Recent work on the widespread crAssphage suggests it might demonstrate long-term associations with its superhosts (7), similar to patterns described for many bacteria (8, 9).Primates host distinct bacterial communities, such that more phylogenetically related host taxa have more similar gut microbial composition (8, 10). The structure of these communities thus recapitulates the host phylogeny [i.e., phylosymbiosis (8, 10)], potentially reflecting widespread cospeciation of bacteria and hosts or phylogenetic conservation of the environments that shape bacterial communities (8, 9). Such long-term host–bacterial associations would imply restricted transmission of bacterial lineages within—rather than between—host lineages (8). This pattern of transmission may be facilitated by the tendency for primates to live in organized societies (11), creating opportunities for bacterial transmission to conspecifics (12, 13). When removed from their natural social and ecological environments and placed in captivity, primates quickly develop humanized bacterial microbiomes (14, 15). This apparent plasticity makes the long-term associations of primates with particular bacterial lineages all the more striking (8, 9).Here, we investigate whether these key findings about primate-associated gut bacterial communities can be generalized to phages. We explore drivers of phage community composition and individual phage lineage evolution in primate superhosts across multiple scales and environments, with a particular emphasis on the potential role of social transmission. We then examine the phageomes of captive primates to understand the flexibility of phage communities in response to the environment and the potential of phage transmission between superhosts. Lastly, we explore whether temperate versus virulent phage lifestyles influence the observed patterns in phage community composition and/or individual phage lineage evolution.     

From the Cover: Overexpression of CD47 is associated with brain overgrowth and 16p11.2 deletion syndrome

Copy number variation (CNV) at the 16p11.2 locus is associated with neuropsychiatric disorders, such as autism spectrum disorder and schizophrenia. CNVs of the 16p gene can manifest in opposing head sizes. Carriers of 16p11.2 deletion tend to have macrocephaly (or brain enlargement), while those with 16p11.2 duplication frequently have microcephaly. Increases in both gray and white matter volume have been observed in brain imaging studies in 16p11.2 deletion carriers with macrocephaly. Here, we use human induced pluripotent stem cells (hiPSCs) derived from controls and subjects with 16p11.2 deletion and 16p11.2 duplication to understand the underlying mechanisms regulating brain overgrowth. To model both gray and white matter, we differentiated patient-derived iPSCs into neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs). In both NPCs and OPCs, we show that CD47 (a “don’t eat me” signal) is overexpressed in the 16p11.2 deletion carriers contributing to reduced phagocytosis both in vitro and in vivo. Furthermore, 16p11.2 deletion NPCs and OPCs up-regulate cell surface expression of calreticulin (a prophagocytic “eat me” signal) and its binding sites, indicating that these cells should be phagocytosed but fail to be eliminated due to elevations in CD47. Treatment of 16p11.2 deletion NPCs and OPCs with an anti-CD47 antibody to block CD47 restores phagocytosis to control levels. While the CD47 pathway is commonly implicated in cancer progression, we document a role for CD47 in psychiatric disorders associated with brain overgrowth.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social interaction and communication. Copy number variation (CNV) at the 16p11.2 locus is associated with ASD (1–8). People who have 16p11.2 deletion syndrome tend to have larger head circumferences (macrocephaly), with disproportionate enlargement in both gray and white matter volume (8–13). Individuals with ASD and macrocephaly have more severe behavioral and cognitive problems and are less responsive to standard medical and therapeutic interventions than those with ASD and normal head circumferences (14). In addition, prior work has documented a very strong cross-sectional and temporal association between macrocephaly and ASD symptoms (8, 9, 11, 12, 14–17). These findings suggest that understanding the underlying mechanisms regulating macrocephaly could provide a window of opportunity for intervention or mitigation of symptoms.Here, we used patient-derived human induced pluripotent stem cells (hiPSCs) to interrogate the underlying mechanisms contributing to gray and white matter enlargement. We focused on individuals with intellectual disability (IQ < 70) or ASD associated with brain overgrowth in 16p11.2 deletion carriers. We differentiated the iPSCs into neural progenitor cells (NPCs) and oligodendrocyte progenitor cells (OPCs) and investigate the hypothesis that brain enlargement in 16p11.2 deletion carriers may be due to improper cellular elimination. Under normal conditions, classic “eat me” and “don’t eat me” signaling mechanisms associated with phagocytosis maintain cellular homeostasis across diverse tissue types (18, 19). CD47 (a “don’t eat me” signal) protects normal cells from getting cleared (18), but can become overexpressed in many types of cancer cells, preventing tumorigenic cells from getting engulfed or phagocytosed (20–22). In fact, CD47 plays an important role in many pathological disorders associated with an overproduction of cells and cell removal, including cancer (20–22), atherosclerosis (23), and fibrotic diseases (24). NPCs derived from iPSCs of autistic individuals with macrocephaly have increased proliferation relative to controls (25, 26). Therefore, we hypothesized that CD47 may be involved in these disorders.We find that CD47 is overexpressed in NPCs and OPCs derived from 16p11.2 deletion carriers, leading to reduced phagocytosis by macrophages and microglia. Furthermore, the 16p11.2 deletion NPCs and OPCs have increased cell surface expression of calreticulin (CRT, a prophagocytic “eat me” signal), indicating that these cells should be eliminated but are not due to high levels of CD47 (27). Importantly, treatment with a CD47 blocking antibody restores phagocytosis of 16p11.2 deletion NPCs and OPCs to control levels, particularly in 16p_del NPCs and OPCs that have increased cell surface expression of CRT, indicating that the changes in phagocytosis are mediated by cell surface expression of CD47. We thus identify a role for CD47 in 16p11.2 deletion syndrome and highlight the potential importance of blocking CD47 to promote clearance of unhealthy NPCs and OPCs in 16p11.2 deletion with brain overgrowth.     

Modulation of immune cell reactivity with cis-binding Siglec agonists

Inflammatory pathologies caused by phagocytes lead to numerous debilitating conditions, including chronic pain and blindness due to age-related macular degeneration. Many members of the sialic acid-binding immunoglobulin-like lectin (Siglec) family are immunoinhibitory receptors whose agonism is an attractive approach for antiinflammatory therapy. Here, we show that synthetic lipid-conjugated glycopolypeptides can insert into cell membranes and engage Siglec receptors in cis, leading to inhibitory signaling. Specifically, we construct a cis-binding agonist of Siglec-9 and show that it modulates mitogen-activated protein kinase (MAPK) signaling in reporter cell lines, immortalized macrophage and microglial cell lines, and primary human macrophages. Thus, these cis-binding agonists of Siglecs present a method for therapeutic suppression of immune cell reactivity.

Sialic acid-binding immunoglobulin (IgG)-like lectins (Siglecs) are a family of immune checkpoint receptors that are on all classes of immune cells (1–5). Siglecs bind various sialoglycan ligands and deliver signals to the immune cells that report on whether the target is healthy or damaged, “self” or “nonself.” Of the 14 human Siglecs, 9 contain cytosolic inhibitory signaling domains. Accordingly, engagement of these inhibitory Siglecs by sialoglycans suppresses the activity of the immune cell, leading to an antiinflammatory effect. In this regard, inhibitory Siglecs have functional parallels with the T cell checkpoint receptors CTLA-4 and PD-1 (6–9). As with these clinically established targets for cancer immune therapy, there has been a recent surge of interest in antagonizing Siglecs to potentiate immune cell reactivity toward cancer (10). Conversely, engagement of Siglecs with agonist antibodies can suppress immune cell reactivity in the context of antiinflammatory therapy. This approach has been explored to achieve B cell suppression in lupus patients by agonism of CD22 (Siglec-2) (11, 12), and to deplete eosinophils for treatment of eosinophilic gastroenteritis by agonism of Siglec-8 (13). Similarly, a CD24 fusion protein has been investigated clinically as a Siglec-10 agonist for both graft-versus-host disease and viral infection (14, 15).Traditionally, Siglec ligands have been studied as functioning in trans, that is, on an adjacent cell (16–18), or as soluble clustering agents (9, 19). In contrast to these mechanisms of action, a growing body of work suggests that cis ligands for Siglecs (i.e., sialoglycans that reside on the same cell membrane) cluster these receptors and maintain a basal level of inhibitory signaling that increases the threshold for immune cell activation. Both Bassik and coworkers (20) and Wyss-Coray and coworkers (21) have linked the depletion of cis Siglec ligands with increased activity of macrophages and microglia, and other studies have shown that a metabolic blockade of sialic acid renders phagocytes more prone to activation (22).Synthetic ligands are a promising class of Siglec agonists (17, 23, 24). Many examples rely on clustering architectures (e.g., sialopolymers, nanoparticles, liposomes) to induce their effect (19, 23–26). Indeed, we have previously used glycopolymers to study the effects of Siglec engagement in trans on natural killer (NK) cell activity (16). We and other researchers have employed glycopolymers (16, 23), glycan-remodeling enzymes (27, 28), chemical inhibitors of glycan biosynthesis (22), and mucin overexpression constructs (29, 30) to modulate the cell-surface levels of Siglec ligands. However, current approaches lack specificity for a given Siglec.We hypothesized that Siglec-specific cis-binding sialoglycans displayed on immune cell surfaces could dampen immune cell activity with potential therapeutic applications. Here we test this notion with the synthesis of membrane-tethered cis-binding agonists of Siglec-9 (Fig. 1). Macrophages and microglia widely express Siglec-9 and are responsible for numerous pathologies including age-related inflammation (31), macular degeneration (32), neural inflammation (33), and chronic obstructive pulmonary disease (34). We designed and developed a lipid-linked glycopolypeptide scaffold bearing glycans that are selective Siglec-9 ligands (pS9L-lipid). We show that pS9L-lipid inserts into macrophage membranes, binds Siglec-9 specifically and in cis, and induces Siglec-9 signaling to suppress macrophage activity. By contrast, a lipid-free soluble analog (pS9L-sol) binds Siglec-9 but does not agonize Siglec-9 or modulate macrophage activity. Membrane-tethered glycopolypeptides are thus a potential therapeutic modality for inhibiting phagocyte activity.Open in a separate windowFig. 1.Lipid-tethered glycopolypeptides cluster and agonize Siglecs in cis on effector cells. (A) Immune cells express activating receptors that stimulate inflammatory signaling. (B) Clustering of Siglec-9 by cis-binding agonists stimulates inhibitory signaling that quenches activation.     

Crystal structure of a far-red–sensing cyanobacteriochrome reveals an atypical bilin conformation and spectral tuning mechanism

Cyanobacteriochromes (CBCRs) are small, linear tetrapyrrole (bilin)-binding photoreceptors in the phytochrome superfamily that regulate diverse light-mediated adaptive processes in cyanobacteria. More spectrally diverse than canonical red/far-red–sensing phytochromes, CBCRs were thought to be restricted to sensing visible and near UV light until recently when several subfamilies with far-red–sensing representatives (frCBCRs) were discovered. Two of these frCBCRs subfamilies have been shown to incorporate bilin precursors with larger pi-conjugated chromophores, while the third frCBCR subfamily uses the same phycocyanobilin precursor found in the bulk of the known CBCRs. To elucidate the molecular basis of far-red light perception by this third frCBCR subfamily, we determined the crystal structure of the far-red–absorbing dark state of one such frCBCR Anacy_2551g3 from Anabaena cylindrica PCC 7122 which exhibits a reversible far-red/orange photocycle. Determined by room temperature serial crystallography and cryocrystallography, the refined 2.7-Å structure reveals an unusual all-Z,syn configuration of the phycocyanobilin (PCB) chromophore that is considerably less extended than those of previously characterized red-light sensors in the phytochrome superfamily. Based on structural and spectroscopic comparisons with other bilin-binding proteins together with site-directed mutagenesis data, our studies reveal protein–chromophore interactions that are critical for the atypical bathochromic shift. Based on these analyses, we propose that far-red absorption in Anacy_2551g3 is the result of the additive effect of two distinct red-shift mechanisms involving cationic bilin lactim tautomers stabilized by a constrained all-Z,syn conformation and specific interactions with a highly conserved anionic residue.

Cyanobacteria have developed elaborate, spectrally tuned photoreceptors and light-harvesting systems for adaptation and survival in a wide range of ecological niches (1–5). Many photoreceptor systems are modular components of much larger signaling proteins that integrate different sensor and effector modules into a single protein molecule to interface with diverse signal transduction pathways. Photoreceptors in the phytochrome superfamily utilize a specific lineage of GAF (cGMP phosphodiesterase, adenylyl cyclase and FhlA) domain that binds a thioether-linked linear tetrapyrrole (bilin) chromophore for light perception (6–11). Bilin-based photoreceptors play critical roles in plant development as well as in regulating cyanobacterial phototaxis, development, and light harvesting (2, 3, 12–17). Protein structural changes following the primary photochemical event then alter the downstream enzymatic activities and/or protein–protein interactions via an interdomain allosteric mechanism (18).Phytochromes possess a tripartite photosensory region consisting of three N-terminal domains (PAS, GAF, and PHY), known as the photosensory core module, in which the PAS and GAF domains are tethered via a “figure-eight knot” (14, 19, 20). In prototypical phytochromes, the bilin chromophore embedded in the GAF domain adopts a protonated 5-Z,syn, 10-Z,syn, 15-Z,anti configuration in the dark-adapted state. Light absorption triggers photoisomerization of the 15,16 double bond to generate a 15E,anti photoproduct, which typically absorbs far-red light (9, 14, 21). A long extension from the adjacent PHY domain is responsible for stabilizing the far-red–absorbing Pfr state (14, 20). In cyanobacteria, the phytochrome superfamily has diversified to yield a large family of more streamlined sensors, designated cyanobacteriochromes (CBCRs) (2, 4, 22–26). Unlike canonical phytochromes, CBCR photosensory modules consist of one or more GAF domains that are sufficient for covalent attachment of bilin and photoconversion. These small CBCR domains have also been used as light-sensing modules in a variety of synthetic biology applications (27–32). In contrast to canonical red/far-red phytochromes, CBCRs are able to sense light from near UV to far-red, utilizing a common phycocyanobilin (PCB) chromophore precursor (22–24, 26).The remarkable spectral diversity of CBCRs (SI Appendix, Fig. S1A) arises from extensive molecular evolution of the GAF domain scaffold. Many CBCRs leverage two thioether linkages to sense blue, violet, or near-UV light (8, 22, 23, 25, 33–35). Such “two-Cys” CBCRs possess an additional thioether linkage to the C10 methine bridge of the bilin that splits the chromophore in half, significantly shortening the conjugated π-system. Rupture of this covalent bond can occur upon 15Z/15E photoisomerization, which restores bilin conjugation across C10 to generate a photostate absorbing at wavelengths from teal to red (8, 33, 36, 37). Dual cysteine CBCRs have evolved multiple times, yielding a wide range of photocycles with (ultra)violet, blue, teal, green, orange, and red states (22).Red/green CBCRs such as AnPixJg2 and NpR6012g4 have red-absorbing dark states similar to phytochromes that photoconvert to green-absorbing lit states. In this CBCR subfamily, the molecular mechanism responsible for photoproduct tuning relies on trapping the 15E bilin in a twisted geometry that results in blue-shifted absorption (10, 11). In contrast, green/red CBCRs exhibit a reversed photocycle: the green-absorbing 15Z dark state photoconverts to yield a red-absorbing 15E photoproduct. This subfamily uses a protochromic mechanism first reported for the light-regulated histidine kinase RcaE (SI Appendix, Fig. S1B) in which photoconversion triggers a proton transfer to an uncharged chromophore inducing a spectral red shift (2, 38).Until recently, the light-sensing range of CBCRs appeared limited to the visible spectrum, thereby implicating phytochromes to be exclusively responsible for far-red sensing in cyanobacteria. Indeed, far-red–dependent remodeling of the photosynthetic apparatus in multiple cyanobacterial species is mediated by the red/far-red phytochrome RfpA (3, 39). The discovery of two lineages of CBCRs with far-red-absorbing dark states (frCBCRs) was thus surprising (40). Upon far-red light absorption, these frCBCRs convert to either an orange- or red-absorbing photoproduct state. These frCBCRs evolved from green/red CBCRs as part of a greater green/red (GGR) lineage and independent from evolution of other frCBCRs within the XRG (extended red/green) lineage (35, 40, 41). Owing to their small size and spectral overlap with the therapeutic window of optimum tissue penetrance (700 to 800 nm) (42–46), frCBCRs represent tantalizing scaffolds for development of FR-responsive optogenetic reagents for biomedical research and imaging applications (45, 47–50).To understand the molecular basis of far-red spectral tuning of the frCBCR family that evolved within GGR lineage, we determined the crystal structures of the FR-absorbing dark state of the representative FR/O CBCR Anacy_2551g3 from Anabaena cylindrica PCC 7122 at both ambient and cryogenic temperatures. These structures revealed an all-Z,syn configuration of its PCB chromophore that differs from those found in all known CBCRs and phytochromes. Based upon these crystallographic results, spectra of site-directed mutants of Anacy_2551g3 and related frCBCRs in the GGR lineage, and comparisons with other bilin-binding proteins, we identify key protein–chromophore interactions that support two tuning mechanisms simultaneously at work for far-red light detection in this family of frCBCRs.     

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.     

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.     

The evolution of red color vision is linked to coordinated rhodopsin tuning in lycaenid butterflies

Color vision has evolved multiple times in both vertebrates and invertebrates and is largely determined by the number and variation in spectral sensitivities of distinct opsin subclasses. However, because of the difficulty of expressing long-wavelength (LW) invertebrate opsins in vitro, our understanding of the molecular basis of functional shifts in opsin spectral sensitivities has been biased toward research primarily in vertebrates. This has restricted our ability to address whether invertebrate G_q protein-coupled opsins function in a novel or convergent way compared to vertebrate G_t opsins. Here we develop a robust heterologous expression system to purify invertebrate rhodopsins, identify specific amino acid changes responsible for adaptive spectral tuning, and pinpoint how molecular variation in invertebrate opsins underlie wavelength sensitivity shifts that enhance visual perception. By combining functional and optophysiological approaches, we disentangle the relative contributions of lateral filtering pigments from red-shifted LW and blue short-wavelength opsins expressed in distinct photoreceptor cells of individual ommatidia. We use in situ hybridization to visualize six ommatidial classes in the compound eye of a lycaenid butterfly with a four-opsin visual system. We show experimentally that certain key tuning residues underlying green spectral shifts in blue opsin paralogs have evolved repeatedly among short-wavelength opsin lineages. Taken together, our results demonstrate the interplay between regulatory and adaptive evolution at multiple G_q opsin loci, as well as how coordinated spectral shifts in LW and blue opsins can act together to enhance insect spectral sensitivity at blue and red wavelengths for visual performance adaptation.

Opsins belong to a diverse multigene family of G protein-coupled receptors that bind to a small nonprotein retinal moiety to form photosensitive rhodopsins and enable vision across animals (1–4). The tight relationship between opsin genotypes and spectral sensitivity phenotypes offers an ideal framework to analyze how specific molecular changes give rise to adaptations in visual behaviors (5). Notably, independent opsin gene gains and losses (6–13), genetic variation across opsins (14–16), spectral tuning mutations within opsins (17–21), and alterations in visual regulatory networks (22, 23) have contributed to opsin adaptation. Yet, the molecular and structural changes underlying the remarkable diversification of spectral sensitivity phenotypes identified in some invertebrates, including crustaceans and insects (24–27), are far less understood than those in vertebrate lineages (28–32).The diversity of opsin-based photoreceptors observed across animal visual systems is produced by distinct ciliary vertebrate c-opsin and invertebrate rhabdomeric based r-opsin subfamilies that mediate separate phototransduction cascades (31, 33–35). Vertebrate c-opsins function through the G protein transducing (G_t) signaling pathway, which activates cyclic nucleotide phosphodiesterase, ultimately resulting in a hyperpolarization response in photoreceptor cells through the opening of selective K⁺ channels (31, 36). By contrast, insect opsins transmit light stimuli through a G_q-type G protein (33, 37) with phosphoinositol (PLCβ) acting as an effector enzyme to achieve TRP channel depolarization in the invertebrate photoreceptor cell (34, 38).All vertebrate visual cone opsins derive from four gene families: short-wavelength-sensitive opsins SWS1 (or ultraviolet [UV]) with λ_max 344 to 445 nm and SWS2 with λ_max 400 to 470 nm, and longer-wavelength-sensitive opsins that specify the green MWS (or Rh2) pigments with λ_max 480 to 530 nm and red-sensitive LWS pigments with λ_max 500 to 570 nm (5, 30). Most birds and fish have retained the four ancestral opsin genes (39), with notable opsin expansions in cichlid fish opsins (23, 40), whereas SWS1 is extinct in monotremes, and SWS2 and M opsins are lost in marsupials and eutherian mammals (41). In primates, trichromatic vision is conferred through SWS1 (λ_max = 414 nm) and recent duplicate MWS (λ_max = 530 nm) and LWS opsins (λ_max = 560 nm) (42–44). In vertebrates, molecular evolutionary approaches and well-established in vitro opsin purification have identified the complex interplay between opsin duplications, regulatory and protein-coding mutations controlling opsin gene tuning, and spectral phenotypes notably in birds, fish, and mammals (45–47).Insect opsins are phylogenetically distinct but functionally analogous to those of vertebrates, and the ancestral opsin repertoire consists of three types of light-absorbing rhabdomeric G_q-type opsin specifying UV (350 nm), short-wavelength (blue, 440 nm) and long-wavelength pigments (LW, 530 nm) (48). Given the importance of color-guided behaviors and the remarkable photoreceptor spectral diversity observed in insects (26, 27), the dynamic opsin gene diversification found across lineages (Fig. 1) highlights their potentially central role in adaptation (27, 49, 50), yet the molecular basis of opsin functionality of rhabdomeric invertebrate G_q opsins remains understudied.Open in a separate windowFig. 1.Visual opsin gene evolution and spectral tuning mechanisms in insects. Visual opsin genes of the Atala hairstreak (E. atala, Lepidoptera, Lycaenidae) in comparison with those encoded in the genomes of diverse insects. The opsin types are highlighted in gray for UV, in blue for short wavelength (SW), and in green for long wavelength (LW). Numbers indicate multiple opsins, whereas no dot indicates gene loss. Colored circles indicate instances of shifted spectral sensitivities in at least one of the encoded opsins. The direction of shift is inferred from the opsin lambda max that departs from the typical range of absorbance in the opsin subfamily using wavelength boundaries for the various colors: UV <380 nm, violet 380 to 435 nm, blue 435 to 492 nm, green 492 to 530 nm, and red shifted >530 nm. Coleopteran lineages, and some hemipterans, lost the blue opsin locus and compensated for the loss of blue sensitivity via UV and/or LW gene duplications across lineages (11, 12). In butterflies, extended photosensitivity at short wavelengths is observed in Heliconius erato with two UV opsins at λ_max = 355 nm and 398 nm (10) and in P. rapae with two blue opsins with λ_max = 420 and 450 nm (17). A blue opsin duplication occurred independently in lycaenid butterflies (61). LW opsin duplications occurred independently in most major insect lineages (6, 16, 55) and confer a variable range of LW sensitivities with or without additional contributions from lateral filtering. In order to extend spectral sensitivity at longer wavelengths while sharpening blue acuity, some lycaenid butterflies have evolved a new color vision mechanism combining spectral shifts at a duplicate blue opsin and at the LW opsin. Images credit: Christopher Adams (illustrator).The recurrent evolution of red receptors in insects in particular suggests that perception of longer wavelengths can play an important role in the context of foraging, oviposition, and/or conspecific recognition (6, 27, 51–54). In butterflies, several mechanisms are likely to have provided extended spectral sensitivity to longer wavelengths. LW opsin duplications along with the evolution of lateral filtering between ommatidia has been demonstrated in two papilionids, Papilio xuthus (27) and Graphium sarpedon (55), as well as in a riodinid (Apodemia mormo) (6, 54). Lateral filtering pigments are relatively widespread across butterfly lineages, e.g., Heliconius (56), Pieris (57), Colias erate (58), and some moths [Adoxophyes orana (59) and Paysandisia archon (60)]. These pigments absorb short wavelengths and aid in shifting the sensitivity peak of green LW photoreceptors to longer wavelengths (27, 51, 56, 57, 61, 62). Despite creating distinct spectral types that can contribute to color vision, as identified in nymphalid (56), pierid (57), and lycaenid (62) species, all of which lack duplicated LW opsins (61, 63), lateral filtering alone cannot extend photoreceptor sensitivity toward the far red (700 to 750 nm) beyond the exponentially decaying long-wavelength rhodopsin absorbance spectrum (51). Thus, molecular variation of ancestral LW opsin genes is likely to have contributed an as yet underexplored mechanism to the diversification of long-wavelength photoreceptor spectral sensitivity. However, disentangling the relative contributions of lateral filtering and pure LW opsin properties has remained technically challenging using classical electrophysiological approaches (14, 64, although see, e.g., refs. 65, 66, 67) and has been limited by the lack of in vitro expression systems suitable for LW opsins.While opsin duplicates have been identified in numerous organisms, the spectral tuning mechanisms and interplay between new opsin photoreceptors in invertebrate visual system evolution are less well understood. Here we combine physiological, molecular, and heterologous approaches to start closing this gap in our knowledge of invertebrate G_q opsin evolution by investigating the functions, spectral tuning, and implications of evolving new combinations of short- and long-wavelength opsin types in lycaenid species. This butterfly group, comprising the famous blues, coppers, and hairstreaks, is the second largest family with about 5,200 (28%) of the some 18,770 described butterfly species (68). In light of their remarkable behavioral, ecological, and morphological diversity (69, 70), as well as pioneer studies in the Lycaena and Polyommatus genera supporting the rapid evolution of color vision in certain lineages (56, 61, 62), lycaenids provide an ideal candidate system for investigating opsin evolution and visual adaptations. Using the Atala hairstreak, Eumaeus atala, as a molecular and ecological model, we find coordinated spectral shifts at short- and long-wavelength G_q opsin loci and demonstrate that the combination of six ommatidial classes of photoreceptors in the compound eye uniquely extend spectral sensitivity at long wavelengths toward the far-red while concurrently sharpening acuity of multiple blue wavelengths. Together, these findings link the evolution of four-opsin visual systems to adaptation in the context of finely tuned color perception critical to the behavior of these butterflies.