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.     

Photosynthesis tunes quantum-mechanical mixing of electronic and vibrational states to steer exciton energy transfer

Photosynthetic species evolved to protect their light-harvesting apparatus from photoxidative damage driven by intracellular redox conditions or environmental conditions. The Fenna–Matthews–Olson (FMO) pigment–protein complex from green sulfur bacteria exhibits redox-dependent quenching behavior partially due to two internal cysteine residues. Here, we show evidence that a photosynthetic complex exploits the quantum mechanics of vibronic mixing to activate an oxidative photoprotective mechanism. We use two-dimensional electronic spectroscopy (2DES) to capture energy transfer dynamics in wild-type and cysteine-deficient FMO mutant proteins under both reducing and oxidizing conditions. Under reducing conditions, we find equal energy transfer through the exciton 4–1 and 4–2-1 pathways because the exciton 4–1 energy gap is vibronically coupled with a bacteriochlorophyll-a vibrational mode. Under oxidizing conditions, however, the resonance of the exciton 4–1 energy gap is detuned from the vibrational mode, causing excitons to preferentially steer through the indirect 4–2-1 pathway to increase the likelihood of exciton quenching. We use a Redfield model to show that the complex achieves this effect by tuning the site III energy via the redox state of its internal cysteine residues. This result shows how pigment–protein complexes exploit the quantum mechanics of vibronic coupling to steer energy transfer.

Photosynthetic organisms convert solar photons into chemical energy by taking advantage of the quantum mechanical nature of their molecular systems and the chemistry of their environment (1–4). Antenna complexes, composed of one or more pigment–protein complexes, facilitate the first steps in the photosynthesis process: They absorb photons and determine which proportion of excitations to move to reaction centers, where charge separation occurs (4). In oxic environments, excitations can generate highly reactive singlet oxygen species. These pigment–protein complexes can quench excess excitations in these environments with molecular moieties such as quinones and cysteine residues (1, 5–7).The Fenna–Matthews–Olson (FMO) complex, a trimer of pigment–protein complexes found in the green sulfur bacterium Chlorobaculum tepidum (8), has emerged as a model system to study the photophysical properties of photosynthetic antenna complexes (9–19). Each subunit in the FMO complex contains eight bacteriochlorophyll-a site molecules (Protein Data Bank, ID code: 3ENI) that are coupled to form a basis of eight partially delocalized excited states called excitons (Fig. 1) (20–23). Previous experiments on FMO have observed the presence of long-lived coherences in nonlinear spectroscopic signals at both cryogenic and physiological temperatures (11, 13). The coherent signals are thought to arise from some combination of electronic (24–26), vibrational (16–18), and vibronic (27) coherences in the system (28–30). One previous study reported that the coherent signals in FMO remain unchanged upon mutagenesis of the protein, suggesting that the signals are ground state vibrational coherences (17). Others discuss the role of vibronic coupling, where electronic and nuclear degrees of freedom become coupled (29). Other dimeric model systems have demonstrated the regimes in which these vibronically coupled states produce coherent or incoherent transport and vibronic coherences (31–33). Recent spectroscopic data has suggested that vibronic coupling plays a role in driving efficient energy transfer through photosynthetic complexes (27, 31, 33, 34), but to date there is no direct experimental evidence suggesting that biological systems use vibronic coupling as part of their biological function.Open in a separate windowFig. 1.(Left) Numbered sites and sidechains of cysteines C353 and C49 in the FMO pigment–protein complex (PDB ID code: 3ENI) (20). (Right) Site densities for excitons 4, 2, and 1 in reducing conditions with the energy transfer branching ratios for the WT oxidized and reduced protein. The saturation of pigments in each exciton denotes the relative contribution number to the exciton. The C353 residue is located near excitons 4 and 2, which have most electron density along one side of the complex, and other redox-active residues such as the Trp/Tyr chain. C353 and C49 surround site III, which contains the majority of exciton 1 density. Excitons 2 and 4 are generally delocalized over sites IV, V, and VII.It has been shown that redox conditions affect excited state properties in pigment-protein complexes, yet little is known about the underlying microscopic mechanisms for these effects (1, 9). Many commonly studied light-harvesting complexes—including the FMO complex (20), light-harvesting complex 2 (LH2) (35), the PC645 phycobiliprotein (36), and the cyanobacterial antenna complex isiA (37)—contain redox-active cysteine residues in close proximity to their chromophores. As the natural low light environment of C. tepidum does not necessitate photoprotective responses to light quantity and quality, its primary photoprotective mechanism concerns its response to oxidative stress. C. tepidum is an obligate anaerobe, but the presence of many active anoxygenic genes such as sodB for superoxide dismutase and roo for rubredoxin oxygen oxidoreductase (38) suggests that it is frequently exposed to molecular oxygen (7, 39). Using time-resolved fluorescence measurements, Orf et al. demonstrated that two cysteine residues in the FMO complex, C49 and C353, quench excitons under oxidizing conditions (1), which could protect the excitation from generating reactive oxygen species (7, 40–42). In two-dimensional electronic spectroscopy (2DES) experiments, Allodi et al. showed that redox conditions in both the wild-type and C49A/C353A double-mutant proteins affect the ultrafast dynamics through the FMO complex (9, 43). The recent discovery that many proteins across the evolutionary landscape possess chains of tryptophan and tyrosine residues provides evidence that these redox-active residues may link the internal protein behavior with the chemistry of the surrounding environment (41, 43).In this paper, we present data showing that pigment–protein complexes tune the vibronic coupling of their chromophores and that the absence of this vibronic coupling activates an oxidative photoprotective mechanism. We use 2DES to show that a pair of cysteine residues in FMO, C49 and C353, can steer excitations toward quenching sites in oxic environments. The measured reaction rate constants demonstrate unusual nonmonotonic behavior. We then use a Redfield model to determine how the exciton energy transfer (EET) time constants arise from changing chlorophyll site energies and their system-bath couplings (44, 45). The analysis reveals that the cysteine residues tune the resonance between exciton 4–1 energy gap and an intramolecular chlorophyll vibration in reducing conditions to induce vibronic coupling and detune the resonance in oxidizing conditions. This redox-dependent modulation of the vibronic coupling steers excitations through different pathways in the complex to change the likelihood that they interact with exciton quenchers.     

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.     

Replisome bypass of a protein-based R-loop block by Pif1

Efficient and faithful replication of the genome is essential to maintain genome stability. Replication is carried out by a multiprotein complex called the replisome, which encounters numerous obstacles to its progression. Failure to bypass these obstacles results in genome instability and may facilitate errors leading to disease. Cells use accessory helicases that help the replisome bypass difficult barriers. All eukaryotes contain the accessory helicase Pif1, which tracks in a 5′–3′ direction on single-stranded DNA and plays a role in genome maintenance processes. Here, we reveal a previously unknown role for Pif1 in replication barrier bypass. We use an in vitro reconstituted Saccharomyces cerevisiae replisome to demonstrate that Pif1 enables the replisome to bypass an inactive (i.e., dead) Cas9 (dCas9) R-loop barrier. Interestingly, dCas9 R-loops targeted to either strand are bypassed with similar efficiency. Furthermore, we employed a single-molecule fluorescence visualization technique to show that Pif1 facilitates this bypass by enabling the simultaneous removal of the dCas9 protein and the R-loop. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.

Efficient and faithful replication of the genome is essential to maintain genome stability and is carried out by a multiprotein complex called the replisome (1–4). There are numerous obstacles to progression of the replisome during the process of chromosome duplication. These obstacles include RNA-DNA hybrids (R-loops), DNA secondary structures, transcribing RNA polymerases, and other tightly bound proteins (5–9). Failure to bypass these barriers may result in genome instability, which can lead to cellular abnormalities and genetic disease. Cells contain various accessory helicases that help the replisome bypass these difficult barriers (10–20). A subset of these helicases act on the opposite strand of the replicative helicase (1, 2, 14, 19).All eukaryotes contain an accessory helicase, Pif1, which tracks in a 5′–3′ direction on single-stranded DNA (ssDNA) (11–16). Pif1 is important in pathways such as Okazaki-fragment processing and break-induced repair that require the removal of DNA-binding proteins as well as potential displacement of R-loops (11–13, 21, 15–18, 22–25). Genetic studies and immunoprecipitation pull-down assays indicate that Pif1 interacts with PCNA (the DNA sliding clamp), Pol ε (the leading-strand polymerase), the MCMs (the motor subunits of the replicative helicase CMG), and RPA (the single-stranded DNA-binding protein) (15, 26, 27). Pif1 activity in break-induced repair strongly depends on its interaction with PCNA (26). These interactions with replisomal components suggest that Pif1 could interact with the replisome during replication. In Escherichia coli, the replicative helicase is the DnaB homohexamer that encircles the lagging strand and moves in a 5′–3′ direction (20). E. coli accessory helicases include the monomeric UvrD (helicase II) and Rep, which move in the 3′–5′ direction and operate on the opposite strand from the DnaB hexamer. It is known that these monomeric helicases promote the bypass of barriers during replication such as stalled RNA polymerases (5). The eukaryotic replicative helicase is the 11-subunit CMG (Cdc45, Mcm2–7, GINS) and tracks in the 3′–5′ direction, opposite to the direction of Pif1 (25, 28). Once activated by Mcm10, the MCM motor domains of CMG encircle the leading strand (29–32). We hypothesized that, similar to UvrD and Rep in E. coli, Pif1 interacts with the replisome tracking in the opposite direction to enable bypass of replication obstacles.In this report, we use an in vitro reconstituted Saccharomyces cerevisiae replisome to study the role of Pif1 in bypass of a “dead” Cas9 (dCas9), which is a Cas9 protein that is deactivated in DNA cleavage but otherwise fully functional in DNA binding. As with Cas9, dCas9 is a single-turnover enzyme that can be programmed with a guide RNA (gRNA) to target either strand. The dCas9–gRNA complex forms a roadblock consisting of an R-loop and a tightly bound protein (dCas9), a construct that is similar to a stalled RNA polymerase. This roadblock (hereafter dCas9 R-loop) arrests replisomes independent of whether the dCas9 R-loop is targeted to the leading or lagging strand (30). Besides its utility due to its programmable nature (33), the use of the dCas9 R-loop allows us to answer several mechanistic questions. For example, the ability to program the dCas9 R-loop block to any specific sequence enables us to observe whether block removal is different depending on whether the block is on the leading or lagging strand. Furthermore, the inner diameter of CMG can accommodate double-stranded DNA (dsDNA) and possibly an R-loop, but not a dCas9 protein. Using the dCas9 R-loop block allows us to determine the fate of each of its components.Here, we report that Pif1 enables the bypass of the dCas9 R-loop by the replisome. Interestingly, dCas9 R-loops targeted to either the leading or lagging strand are bypassed with similar efficiency. In addition, the PCNA clamp is not required for bypass of the block, indicating that Pif1 does not need to interact with PCNA during bypass of the block. We used a single-molecule fluorescence imaging to show that both the dCas9 and the R-loop are displaced as an intact nucleoprotein complex. We propose that Pif1 is a general displacement helicase for replication bypass of both R-loops and protein blocks.     

Optical vector field rotation and switching with near-unity transmission by fully developed chiral photonic crystals

State-of-the-art nanostructured chiral photonic crystals (CPCs), metamaterials, and metasurfaces have shown giant optical rotatory power but are generally passive and beset with large optical losses and with inadequate performance due to limited size/interaction length and narrow operation bandwidth. In this work, we demonstrate by detailed theoretical modeling and experiments that a fully developed CPC, one for which the number of unit cells N is high enough that it acquires the full potentials of an ideal (N → ∞) crystal, will overcome the aforementioned limitations, leading to a new generation of versatile high-performance polarization manipulation optics. Such high-N CPCs are realized by field-assisted self-assembly of cholesteric liquid crystals to unprecedented thicknesses not possible with any other means. Characterization studies show that high-N CPCs exhibit broad transmission maxima accompanied by giant rotatory power, thereby enabling large (>π) polarization rotation with near-unity transmission over a large operation bandwidth. Polarization rotation is demonstrated to be independent of input polarization orientation and applies equally well on continuous-wave or ultrafast (picosecond to femtosecond) pulsed lasers of simple or complex (radial, azimuthal) vector fields. Liquid crystal–based CPCs also allow very wide tuning of the operation spectral range and dynamic polarization switching and control possibilities by virtue of several stimuli-induced index or birefringence changing mechanisms.

Optical vector field (more commonly called polarization) rotators and switches are essential components of all modern optical and photonic systems for communications, ellipsometry, metrology, biological/chemical detection, and quantum processing/computing (1–10). There are, however, some inherent limitations. Wave plates made with birefringent crystals, for example, require strict alignment of the optic axis with respect to the polarization orientation of incident light and generally do not work with laser vector beams of complex polarization fields; Faraday rotators that do not have this requirement are generally too cumbersome and bulky due to their weak optical rotatory powers. One promising approach to circumvent these limitations is to employ chiral optical materials such as chiral photonic crystals and metasurfaces. Nevertheless, structural chirality, such as chiral metamaterials, metasurfaces, and photonic crystals that are capable of very large optical rotatory power (up to ∼100,000°/mm), are inevitably accompanied by large absorption losses (11–15). In metamaterials/surfaces, the intrinsic noncircular absorption and nanofabrication difficulty also add to the limitation of their practical scalability in the interaction length, resulting in small (<π) net polarization rotation angle, very small aperture, and narrow operating spectral bandwidth (11–13). Similar issues confront most chiral photonic crystals (CPCs) due to the limitations of molecular self-assembly or nanofabrication/processing technique and high transmission loss associated with operation near the Bragg reflection band (14, 15).Here, we show by theory and experimental corroborations that a fully developed liquid crystal–based CPC, one for which the number of unit cells N approaches that (N → ∞) of an ideal crystal, can circumvent all the aforementioned limitations and possess several advantageous characteristics impossible with conventional low-N thin counterparts. Such high-period–number chiral photonic crystals (HN-CPCs) are achieved by fabricating cholesteric liquid crystals (CLCs) to thicknesses several hundred times that of conventional ones using a refined field-assisted self-assembly (FASA) technique (16, 17; see SI Appendix, Note 1, for more details). Optical properties of CLCs as CPCs arise from complex “collective” responses from many unit cells. While thicker crystals obviously give rise to larger effects, the resulting properties as the crystal thickness or period number N evolves from low values to a very high value do not lend themselves to such simple linear extrapolation; as a function of N, pleasant surprises and new insights and possibilities abound. Our studies show that for N > 500, these CLCs exhibit simultaneously broad transmission maxima and large polarization rotation power in the off-Bragg-resonance spectral regime. Polarization rotation is independent of input polarization orientation and acts equally well on simple or complex vector fields (18–22) of continuous-wave (CW) or ultrafast pulsed laser beams. Liquid crystal–based CPCs also allow dynamic polarization switching and control by virtue of field–induced index/birefringence changing mechanisms at modest or ultrafast (picosecond to femtosecond) speeds (23–34).     

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.     

Interplays of electron and nuclear motions along CO dissociation trajectory in myoglobin revealed by ultrafast X-rays and quantum dynamics calculations

Ultrafast structural dynamics with different spatial and temporal scales were investigated during photodissociation of carbon monoxide (CO) from iron(II)–heme in bovine myoglobin during the first 3 ps following laser excitation. We used simultaneous X-ray transient absorption (XTA) spectroscopy and X-ray transient solution scattering (XSS) at an X-ray free electron laser source with a time resolution of 80 fs. Kinetic traces at different characteristic X-ray energies were collected to give a global picture of the multistep pathway in the photodissociation of CO from heme. In order to extract the reaction coordinates along different directions of the CO departure, XTA data were collected with parallel and perpendicular relative polarizations of the laser pump and X-ray probe pulse to isolate the contributions of electronic spin state transition, bond breaking, and heme macrocycle nuclear relaxation. The time evolution of the iron K-edge X-ray absorption near edge structure (XANES) features along the two major photochemical reaction coordinates, i.e., the iron(II)–CO bond elongation and the heme macrocycle doming relaxation were modeled by time-dependent density functional theory calculations. Combined results from the experiments and computations reveal insight into interplays between the nuclear and electronic structural dynamics along the CO photodissociation trajectory. Time-resolved small-angle X-ray scattering data during the same process are also simultaneously collected, which show that the local CO dissociation causes a protein quake propagating on different spatial and temporal scales. These studies are important for understanding gas transport and protein deligation processes and shed light on the interplay of active site conformational changes and large-scale protein reorganization.

Enzymatic functions frequently involve local motions at the active site as well as large-amplitude motions of the protein, and the two are often strongly correlated. Many chemical processes at the active sites take place as a result of the interplay between atomic movement and electronic structural changes in response to external stimuli such as light, ligand binding, heat or electric field. While reaction kinetics can be predicted from thermodynamic properties, the intrinsic time scales for fundamental chemical events, such as bond breakage and formation, are often unresolved due to challenges in examining rapid electronic and atomic movements in real time. Advanced X-ray sources, especially those with intense photon bursts within the time scale of fundamental chemical events (i.e., femtoseconds), enable structural characterization in terms of the electronic and atomic motions. Combining such ultrashort X-ray pulses with laser excitation, we are able to detect the interplay of ultrafast electron and nuclear motions in the photodissociation of an axial CO ligand from the iron center in the heme site of myoglobin (Mb) (Fig. 1). The same process has been extensively studied due to numerous functions of heme or other iron porphyrins in hemoproteins, including electron transfer, catalytic oxidation or reduction of metabolites, neutralization of damaging reactive species, and famously the binding of diatomics such as dioxygen, carbon monoxide, and nitric oxide for transportation and sensing (1–6). Because the dissociation of diatomic ligands, such as CO and NO, can be synchronized through optical excitation of the porphyrin, diatomic ligand binding in hemoproteins is amenable to scrutiny by dynamic structural and electronic spectroscopies (1, 7–14). Several X-ray diffraction, solution scattering, and X-ray spectroscopy (including X-ray absorption and emission) studies have been carried out using intense X-ray pulses from synchrotron and X-ray free electron laser sources (11, 14–19). In this report, we focus on the correlations between the electronic structural change of the iron center and these nuclear motions. To investigate these correlations, we used X-ray transient absorption spectroscopy/scattering and theoretical calculations to detect and project detailed trajectories for the CO departure from Fe(II) in the heme site of bovine Mb.Open in a separate windowFig. 1.(A) Mb structure with heme in pink surrounded by helices of the protein. (B) Mb active site structural changes following CO photolysis. Upon photoexcitation, ground state MbCO (green) loses its bond to CO and adopts a square pyramidal structure with His93 (pink), resulting in the doming of the porphyrin where the Fe (red) comes out of the plane of the macrocycle. Structures are from photolysed MbCO trapped at low temperature (12) and ground state (9) MbCO, where their crystal structures are aligned by their respective porphyrin carbons.In carbonmonoxymyoglobin (MbCO), the low-spin (LS) Fe(II) center has a pseudo-octahedral coordination geometry, ligated with four nitrogens (N_p) from the heme, the nitrogen of an axial histidine (N_His, His93), and CO, a strong field ligand. Previous studies have pointed out that upon excitation of the heme Soret or Q band, photolysis occurs within ∼50 fs, although there is an ongoing debate about the mechanism of CO photodissociation and the subsequent relaxation of the heme, as well as the possible role of intermediate spin states, similar to those observed in photoexcited iron Tris(bipyridine) (20) and ferrous cytochrome c (14, 15, 21). With the loss of CO, the LS state of Fe(II) transforms to a high-spin (HS) state and adopts square-pyramidal pentacoordination with the axial histidine His93 moving ∼0.3 Å out of the porphyrin plane, perturbing the position of the alpha helix in which it sits (Fig. 1B) (4, 8, 22, 23). Protein control of this movement is critical, both because it is the first step of the mechanism of cooperativity in hemoglobin O₂ binding and because it may lead to a conformational rearrangement of the heme pocket that allows CO to escape and avoid geminate recombination (24).This CO photodissociation from MbCO, as well as the photolysis of other diatomics such as NO, and the subsequent recombination dynamics have been assessed using X-ray transient absorption (XTA) spectroscopy (Fig. 2) at synchrotron sources with ∼100-ps time resolution (19, 25), using Fe K-edge X-ray absorption near edge structure (XANES) spectral features shown in Fig. 2. The main differences between the spectral features of MbCO and Mb are an edge shift to a lower energy and a preedge conversion from two sharp peaks to one broader and weaker peak. These changes are consistent with loss of CO and a conversion of Fe(II) from LS to HS in Mb, as supported by optical and vibrational spectroscopic studies (8, 26).Open in a separate windowFig. 2.Fe K-edge XANES and difference spectra measured after CO photodissociation with 100 ps time resolution (19). Energies selected for measurement of polarization-dependent dynamics are marked in dashed vertical cyan lines: line 1, 7.112 keV, the depletion of the ground state transition of 1s → 3d_z2, 3d_x2-y2 character; line 2, 7.115 keV, the disappearance of the preedge peak associated with the CO back bonding antibonding orbital; line 3, 7.118 keV, the rising edge shoulder that appears in MbFe(II); line 4, 7.123 keV, the edge shift; and line 5, 7.172 keV, an EXAFS energy where changes are purely based on changes in the local geometry.While heme vibrational cooling was observed by time-resolved Raman techniques on the time scales of a few to tens of ps (26, 27), and optical transient absorption spectroscopy shows the development of broad excited state absorption features with lifetimes of ∼300 fs and 3 ps, there is an active discussion in the literature as to whether these features can be assigned to an excited-state evolution through a series of electronic intermediate states/species (28–31) or to an exclusively vibrational relaxation pathway (31–33). Because Fe K-edge XTA is sensitive to both the heme iron electronic configuration and the local structural geometry during heme relaxation, measurements of the XANES should distinguish between these mechanisms but only if very fast time scales are resolvable. In this regard, XTA at Linac Coherent Light Source (LCLS) provides a rare opportunity to investigate these relaxation processes with a technique with both high temporal and structural resolution.Although the photodissociation of CO from heme and the concomitant LS to HS transition and heme doming motion are well-known phenomena, many fundamental transformations in terms of electronic and nuclear motions that result in the CO departure are not well understood. The recent works on cytochtome c and NO-bound myoglobin have made progress in the timing of the spin state transitions on the femtosecond time scale and the identification of the intermediate spin state (14, 15). However, it is far from clear how the spin state change was induced electronically, how the iron spin state is related to the Fe–CO distance, and when the heme doming takes place as the Fe–CO distance elongates in dissociation processes. Understanding these correlations has important implications for other chemical and enzymatic processes involving ligand dissociation. It is therefore of great interest to link dynamic structural and electronic changes at the heme during ligand dissociation to more large-scale conformational changes, especially on the time scale of ligand departure and heme doming, which is expected to take place on tens of femtoseconds to a few picoseconds time scales.In order to address the dynamic interplay between electronic and nuclear motions that are beyond the Born–Oppenheimer approximation, we carried out combined XTA and X-ray transient scattering measurements during CO photodissociation with sub–100-fs time resolution at the X-ray Pump-Probe (XPP) facility of the LCLS. The kinetics of the spin state transition and the nuclear motion associated with doming, as well as the global motions of the protein matrix, have revealed a series of correlated events as CO departs from the heme. Although the exact trajectory of CO departure in terms of Fe–CO distance and other structural parameters is still difficult to resolve, such processes can be simulated via quantum mechanical calculations to model this process for the Fe(II) center and the ligands directly bound to the metal. The results provide the energetics of different excited states as well as their trajectories as functions of local structural changes, such as Fe–CO distance and heme relaxation, to distinguish the effect of different structural factors on the overall structural dynamics. These calculations also allow us to predict XTA features without and with the structural relaxation of the heme, providing insight into the interplays between the electronic spin state/configuration and corresponding nuclear motions as a function of the Fe–CO distance.     

LGI1–ADAM22–MAGUK configures transsynaptic nanoalignment for synaptic transmission and epilepsy prevention

Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1–ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv₁ channels, and LRRTM4–Neurexin adhesion molecules. Adam22^ΔC5/ΔC5 knock-in mice devoid of the ADAM22–MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1–ADAM22–MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.

Epilepsy, characterized by unprovoked, recurrent seizures, affects 1 to 2% of the population worldwide. Many genes that cause inherited epilepsy when mutated encode ion channels, and dysregulated synaptic transmission often causes epilepsy (1, 2). Although antiepileptic drugs have mainly targeted ion channels, they are not always effective and have adverse effects. It is therefore important to clarify the detailed processes for synaptic transmission and how they are affected in epilepsy.Recent superresolution imaging of the synapse reveals previously overlooked subsynaptic nano-organizations and pre- and postsynaptic nanodomains (3–6), and mathematical simulation suggests their nanometer-scale coordination in individual synapses for efficient synaptic transmission: presynaptic neurotransmitter release machinery and postsynaptic receptors precisely align across the synaptic cleft to make “transsynaptic nanocolumns” (7, 8).So far, numerous transsynaptic cell-adhesion molecules have been identified (9–12), including presynaptic Neurexins and type IIa receptor protein tyrosine phosphatases (PTPδ, PTPσ, and LAR) and postsynaptic Neuroligins, LRRTMs, NGL-3, IL1RAPL1, Slitrks, and SALMs. Neurexins–Neuroligins have attracted particular attention because of their synaptogenic activities when overexpressed and their genetic association with neuropsychiatric disorders (e.g., autism). Another type of transsynaptic adhesion complex mediated by synaptically secreted Cblns (e.g., Neurexin–Cbln1–GluD2) promotes synapse formation and maintenance (13–15). Genetic studies in Caenorhabditis elegans show that secreted Ce-Punctin, the ortholog of the mammalian ADAMTS-like family, specifies cholinergic versus GABAergic identity of postsynaptic domains and functions as an extracellular synaptic organizer (16). However, the molecular identity and in vivo physiological significance of transsynaptic nanocolumns remain incompletely understood.LGI1, a neuronal secreted protein, and its receptor ADAM22 have recently emerged as major determinants of brain excitability (17) as 1) mutations in the LGI1 gene cause autosomal dominant lateral temporal lobe epilepsy (18); 2) mutations in the ADAM22 gene cause infantile epileptic encephalopathy with intractable seizures and intellectual disability (19, 20); 3) Lgi1 or Adam22 knockout mice display lethal epilepsy (21–24); and 4) autoantibodies against LGI1 cause limbic encephalitis characterized by seizures and amnesia (25–28). Functionally, LGI1–ADAM22 regulates AMPA receptor (AMPAR) and NMDA receptor (NMDAR)-mediated synaptic transmission (17, 22, 29) and Kv₁ channel-mediated neuronal excitability (30, 31). Recent structural analysis shows that LGI1 and ADAM22 form a 2:2 heterotetrameric assembly (ADAM22–LGI1–LGI1–ADAM22) (32), suggesting the transsynaptic configuration.In this study, we identify ADAM22-mediated synaptic protein networks in the brain, including pre- and postsynaptic MAGUKs and their functional bindings to transmembrane proteins (NMDA/AMPA glutamate receptors, voltage-dependent ion channels, cell-adhesion molecules, and vesicle-fusion machinery). ADAM22 knock-in mice lacking the MAGUK-binding motif show lethal epilepsy of hippocampal origin. In this mouse, postsynaptic PSD-95 nano-assembly as well as nano-scale alignment between pre- and postsynaptic proteins are significantly impaired. Importantly, PSD-95 is no longer able to modulate AMPAR-mediated synaptic transmission without binding to ADAM22. These findings establish that LGI1–ADAM22 instructs MAGUKs to organize transsynaptic nanocolumns and guarantee the stable brain activity.     

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.     

Riboflavin instability is a key factor underlying the requirement of a gut microbiota for mosquito development

We previously determined that several diets used to rear Aedes aegypti and other mosquito species support the development of larvae with a gut microbiota but do not support the development of axenic larvae. In contrast, axenic larvae have been shown to develop when fed other diets. To understand the mechanisms underlying this dichotomy, we developed a defined diet that could be manipulated in concert with microbiota composition and environmental conditions. Initial studies showed that axenic larvae could not grow under standard rearing conditions (27 °C, 16-h light: 8-h dark photoperiod) when fed a defined diet but could develop when maintained in darkness. Downstream assays identified riboflavin decay to lumichrome as the key factor that prevented axenic larvae from growing under standard conditions, while gut community members like Escherichia coli rescued development by being able to synthesize riboflavin. Earlier results showed that conventional and gnotobiotic but not axenic larvae exhibit midgut hypoxia under standard rearing conditions, which correlated with activation of several pathways with essential growth functions. In this study, axenic larvae in darkness also exhibited midgut hypoxia and activation of growth signaling but rapidly shifted to midgut normoxia and arrested growth in light, which indicated that gut hypoxia was not due to aerobic respiration by the gut microbiota but did depend on riboflavin that only resident microbes could provide under standard conditions. Overall, our results identify riboflavin provisioning as an essential function for the gut microbiota under most conditions A. aegypti larvae experience in the laboratory and field.

Diet crucially affects the health of all animals (1). Most animals have a gut microbiota that can also affect host health both positively and negatively (2–6). However, understanding of the mechanisms underlying the effects of the gut microbiota remains a major challenge. This is because animals often consume complex or variable diets, and harbor large, multimember microbial communities that can result in many interactions that hinder identification of the factors responsible for particular host responses (2, 6–11). Metaanalyses and multiomic approaches can provide inferential insights on how diet–microbe or microbe–microbe interactions affect hosts (11–18), but functional support can be difficult to generate if proposed mechanisms cannot be studied experimentally (2, 14). Thus, study systems where hosts can be reared on defined diets with or without a microbiota of known composition can significantly advance mechanistic insights by providing the means to control and manipulate dietary, microbial, and environmental variables that potentially affect a given host response (19–21).Mosquitoes are best known as insects that blood feed on humans and other vertebrates. Only adult-stage female mosquitoes blood feed, which is required for egg formation by most species (22). Blood feeding has also led to several mosquitoes evolving into vectors that can transmit disease-causing microbes between hosts (22). In contrast, the juvenile stages of all mosquitoes are aquatic, with most species feeding on detritivorous diets (22–24). Larvae hatch from eggs with no gut microbiota but quickly acquire relatively low-diversity communities from the environment by feeding (25). Most gut community members are aerobic or facultatively anaerobic bacteria in four phyla (Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria), although other microbes, such as fungi and apicomplexans, have also been identified (25–39). Gut community composition also commonly varies within and between species as a function of where larvae develop, diet, and other variables (28–30, 32, 34, 40–42).Aedes aegypti has a worldwide distribution in tropical and subtropical regions, and is the primary vector of the agents that cause yellow fever, dengue fever, and lymphatic filariasis in humans (43). Preferentially living in urban habitats, females lay eggs in water-holding containers with microbial communities, and larvae molt through four instars before pupating and emerging as adults (30, 35, 41, 43). Conventionally reared cultures with a gut microbiota are usually maintained in the laboratory under conditions that mimic natural habitats with rearing temperatures of 25 to 28 °C and a 12- to 16-h light: 8- to 12-h dark photoperiod (44–46). Most insects that require microbial partners for survival live on nutrient-poor diets where microbes provision nutrients that cannot be synthesized or produced in sufficient abundance by the host (3). Mosquito larvae can experience resource limitations in the field (23–25), but in the laboratory are reared on undefined, nutrient-rich diets, such as rodent chow, fish food flakes, or mixtures of materials like liver powder, fish meal, and yeast extract (44–46). Nonetheless, our previous studies indicated that axenic A. aegypti as well as other species consume but fail to grow beyond the first instar when fed several diets that support the development of nonsterile, conventionally reared larvae (30, 47–49). Escherichia coli and several other bacteria identified as gut community members could colonize the gut (producing monoxenic, gnotobiotic larvae) and rescue development, but feeding axenic larvae dead bacteria could not (30, 35, 47). The presence of a gut microbiota in conventional and gnotobiotic but not axenic larvae was also associated with midgut hypoxia and activation of several signaling pathways with growth functions (50, 51). Finally, our own previous results using a strain of E. coli susceptible to ampicillin (50), and more recently a method for clearing an auxotrophic strain of E. coli from gnotobiotic larvae (52), both showed that the proportion of individuals that develop into adults correlates with the duration that larvae have living bacteria in their gut.Altogether, the preceding results suggested that A. aegypti and several other mosquitoes require a gut microbiota for development. In contrast, another recent study showed that axenic A. aegypti larvae develop into adults, albeit more slowly than larvae with a gut microbiota, when fed diets comprised of autoclaved bovine liver powder (LP) and brewer’s yeast (Saccharomyces cerevisiae) extract (YE) or autoclaved LP, YE, and E. coli (EC) embedded in agar (53). This latter finding suggests the undefined dietary components used provide factors larvae require for development into adults, whereas a gut microbiota was also required to provide these factors under the conditions in which our own previous studies were conducted. The goal of this study was to identify what these factors are. Toward this end, we first assessed the growth of axenic A. aegypti when fed diets containing autoclaved LP, YE, and EC under different conditions. We then used this information to develop a defined diet that allowed us to systematically manipulate nutrient, microbial, and environmental variables. We report that the instability of riboflavin is a key factor underlying why A. aegypti larvae require a gut microbiota under most conditions experienced in the laboratory and field.     

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.     

Collagen IV differentially regulates planarian stem cell potency and lineage progression

The extracellular matrix (ECM) provides a precise physical and molecular environment for cell maintenance, self-renewal, and differentiation in the stem cell niche. However, the nature and organization of the ECM niche is not well understood. The adult freshwater planarian Schmidtea mediterranea maintains a large population of multipotent stem cells (neoblasts), presenting an ideal model to study the role of the ECM niche in stem cell regulation. Here we tested the function of 165 planarian homologs of ECM and ECM-related genes in neoblast regulation. We identified the collagen gene family as one with differential effects in promoting or suppressing proliferation of neoblasts. col4-1, encoding a type IV collagen α-chain, had the strongest effect. RNA interference (RNAi) of col4-1 impaired tissue maintenance and regeneration, causing tissue regression. Finally, we provide evidence for an interaction between type IV collagen, the discoidin domain receptor, and neuregulin-7 (NRG-7), which constitutes a mechanism to regulate the balance of symmetric and asymmetric division of neoblasts via the NRG-7/EGFR pathway.

Across the animal kingdom, stem cell function is regulated by the microenvironment in the surrounding niche (1), where the concentration of molecular signals for self-renewal and differentiation can be precisely regulated (2). The niche affects stem cell biology in many processes, such as aging and tissue regeneration, as well as pathological conditions such as cancer (3). Most studies have been done in tissues with large stem cell populations, such as the intestinal crypt (4) and the hair follicle (5) in mice. Elucidation of the role of the stem cell niche in tissue regeneration requires the study of animals with high regenerative potential, such as freshwater planarians (flatworms) (6). Dugesia japonica and Schmidtea mediterranea are two well-studied species that possess the ability to regenerate any missing body part (6, 7).Adult S. mediterranea maintain a high number of stem cells (neoblasts)—∼10 to 30% of all somatic cells in the adult worm—with varying potency, including pluripotent cells (8–14). Neoblasts are the only proliferating somatic cells: they are molecularly heterogeneous, but all express piwi-1 (15–18). Lineage-committed neoblasts are “progenitors” that transiently express both piwi-1 and tissue-specific genes (15, 19). Examples include early intestinal progenitors (γ neoblast, piwi-1⁺/hnf4⁺) (8, 10, 15, 19–21) and early epidermal progenitors (ζ neoblast, piwi-1⁺/zfp-1⁺) (8, 15). Other progenitor markers include collagen for muscles (22), ChAT for neurons (23), and cavII for protonephridia (24, 25). During tissue regeneration, neoblasts are recruited to the wound site, where they proliferate then differentiate to replace the missing cell types (16, 26). Some neoblasts express the pluripotency marker tgs-1, and are designated as clonogenic neoblasts (cNeoblasts) (10, 11). cNeoblasts are located in the parenchymal space adjacent to the gut (11).Neoblasts are sensitive to γ-irradiation and can be preferentially depleted in the adult planarian (27). After sublethal γ-irradiation, remaining cNeoblasts can repopulate the stem cell pool within their niche (10, 11). The close proximity of neoblasts to the gut suggests gut may be a part of neoblast niche (28, 29). When gut integrity was impaired by silencing gata4/5/6, the egfr-1/nrg-1 ligand-receptor pair, or wwp1, maintenance of non–γ-neoblasts were also disrupted (20, 30, 31), but whether that indicates the gut directly regulates neoblast remains unclear. There is evidence indicating the dorsal-ventral (D/V) transverse muscles surrounding the gut may promote neoblast proliferation and migration, with the involvement of matrix metalloproteinase mt-mmpB (32, 33). The central nervous system has also been implicated in influencing neoblast maintenance through the expression of EGF homolog neuregulin-7 (nrg-7), a ligand for EGFR-3, affecting the balance of neoblast self-renewal (symmetric or asymmetric division) (34).In other model systems, an important component of the stem-cell niche is the extracellular matrix (ECM) (35). Germline stem cells in Drosophila are anchored to niche supporting cells with ECM on one side, while the opposite side is exposed to differentiation signals, allowing asymmetric cell fate outcomes for self-renewal or differentiation following division (36–38). Few studies have addressed the ECM in planarians, largely due to the lack of genetic tools to manipulate the genome, the absence of antibodies to specific planarian ECM homologs, or the tools required to study cell fate changes. However, the genomes of D. japonica (39–41) and S. mediterranea (41–45), and single-cell RNA-sequencing (scRNA-seq) datasets for S. mediterranea are now available (11, 46–50). A recent study of the planarian matrisome demonstrated that muscle cells are the primary source of many ECM proteins (51), which, together with those produced by neoblasts and supporting parenchymal cells, may constitute components of the neoblast niche. For example, megf6 and hemicentin restrict neoblast’s localization within the parenchyma (51, 52). Functional studies also implicate ECM-modifiers, such as matrix metalloproteases (MMPs) in neoblast migration and regeneration. For example, reducing the activity of the ECM-degrading enzymes mt-mmpA (26, 33), mt-mmpB (53), or mmp-1 (33) impaired neoblast migration, proliferation, or overall tissue growth, respectively. Neoblasts are also likely to interact with ECM components of the niche via cell surface receptors, such as β1 integrin, inactivation of which impairs brain regeneration (54, 55).Here, we identified planarian ECM homologs in silico, followed by systematic functional assessment of 165 ECM and ECM-related genes by RNA interference (RNAi), to determine the effect on neoblast repopulation in planarians challenged by a sublethal dose of γ-irradiation (10). Surprisingly, multiple classes of collagens were shown to have the strongest effects. In particular, we show that the type IV collagens (COLIV) of basement membranes (BMs), were required to regulate the repopulation of neoblasts as well as lineage progression to progenitor cells. Furthermore, our data support an interaction between COLIV and the discoidin domain receptor (DDR) in neurons that activates signaling of NRG-7 in the neoblasts to regulate neoblast self-renewal versus differentiation. Together, these data demonstrate multifaceted regulation of planarian stem cells by ECM components.     

Microstructural design for mechanical–optical multifunctionality in the exoskeleton of the flower beetle Torynorrhina flammea

Biological systems have a remarkable capability of synthesizing multifunctional materials that are adapted for specific physiological and ecological needs. When exploring structure–function relationships related to multifunctionality in nature, it can be a challenging task to address performance synergies, trade-offs, and the relative importance of different functions in biological materials, which, in turn, can hinder our ability to successfully develop their synthetic bioinspired counterparts. Here, we investigate such relationships between the mechanical and optical properties in a multifunctional biological material found in the highly protective yet conspicuously colored exoskeleton of the flower beetle, Torynorrhina flammea. Combining experimental, computational, and theoretical approaches, we demonstrate that a micropillar-reinforced photonic multilayer in the beetle’s exoskeleton simultaneously enhances mechanical robustness and optical appearance, giving rise to optical damage tolerance. Compared with plain multilayer structures, stiffer vertical micropillars increase stiffness and elastic recovery, restrain the formation of shear bands, and enhance delamination resistance. The micropillars also scatter the reflected light at larger polar angles, enhancing the first optical diffraction order, which makes the reflected color visible from a wider range of viewing angles. The synergistic effect of the improved angular reflectivity and damage localization capability contributes to the optical damage tolerance. Our systematic structural analysis of T. flammea’s different color polymorphs and parametric optical and mechanical modeling further suggest that the beetle’s microarchitecture is optimized toward maximizing the first-order optical diffraction rather than its mechanical stiffness. These findings shed light on material-level design strategies utilized in biological systems for achieving multifunctionality and could thus inform bioinspired material innovations.

Natural selection can act on multiple fronts during the diversification and refinement of morphological characters within a given species (1, 2). These evolutionary processes frequently act both independently and synergistically, resulting in characters that exhibit multifunctionality (3–6). While classical characters, particularly those that have been employed for trait-based taxonomic purposes, typically represent macroscale features such as the size and shapes of bird beaks (7), species-specific characters (or phenotypes) also exist at the “material level” as nano- and microstructures (8–12). These micro- and nanoscale architectures have evolved to enable specific and diverse biological functions including, for example, mechanical protection and optical appearance (10, 13–15). As is the case for macroscale characteristics, a specific microscale material morphology is often the basis for multiple concurrent functionalities (4, 16–19). This observation then raises fundamental questions about multifunctional material design in biology: what types of property-related synergies and trade-offs result from function-specific structural constraints? And for specific multifunctional biological materials, which property or set of properties has gained priority in shaping a specific hierarchical material architecture of interest?Here, we explore functional synergies and trade-offs in a multifunctional biological material system found in the exoskeleton of the flower beetle Torynorrhina flammea. In arthropods, the cuticle forms the hard external “skin” (20), which can simultaneously offer mechanical protection (9, 21), optical signaling (10, 22), water capturing (11), and sensory functions (23). With often incomplete knowledge of the structure–function relationships in biological materials, it is challenging to infer how their microstructures are tailored to fulfill various functional needs, especially considering the fact that this process requires the exploitation of structural synergies and the mitigation of unavoidable compromises in functional performance (3, 24). In all material systems, whether natural or synthetic (4, 25), significant performance trade-offs have to be addressed or accepted when designing for multifunctionality (4, 25–27). As such, the investigation of nature’s strategies for dealing with trade-offs between different properties to achieve multifunctionality is not only of interest to biologists but also to materials scientists and engineers (4–6). Our study of the multifunctional material architecture in the exoskeletons of T. flammea beetles, which enables optical functionality and mechanical robustness simultaneously, provides an example of how to assess functional hierarchy, thereby quantifying the relationship between different material properties in multifunctional material systems. Beyond the analysis of structure–function relationships in biological systems, similar approaches to those described here could also be applied to investigate and optimize the design of engineered multifunctional structural materials.