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

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

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

Entropic formation of a thermodynamically stable colloidal quasicrystal with negligible phason strain

Quasicrystals have been discovered in a variety of materials ranging from metals to polymers. Yet, why and how they form is incompletely understood. In situ transmission electron microscopy of alloy quasicrystal formation in metals suggests an error-and-repair mechanism, whereby quasiperiodic crystals grow imperfectly with phason strain present, and only perfect themselves later into a high-quality quasicrystal with negligible phason strain. The growth mechanism has not been investigated for other types of quasicrystals, such as dendrimeric, polymeric, or colloidal quasicrystals. Soft-matter quasicrystals typically result from entropic, rather than energetic, interactions, and are not usually grown (either in laboratories or in silico) into large-volume quasicrystals. Consequently, it is unknown whether soft-matter quasicrystals form with the high degree of structural quality found in metal alloy quasicrystals. Here, we investigate the entropically driven growth of colloidal dodecagonal quasicrystals (DQCs) via computer simulation of systems of hard tetrahedra, which are simple models for anisotropic colloidal particles that form a quasicrystal. Using a pattern recognition algorithm applied to particle trajectories during DQC growth, we analyze phason strain to follow the evolution of quasiperiodic order. As in alloys, we observe high structural quality; DQCs with low phason strain crystallize directly from the melt and only require minimal further reduction of phason strain. We also observe transformation from a denser approximant to the DQC via continuous phason strain relaxation. Our results demonstrate that soft-matter quasicrystals dominated by entropy can be thermodynamically stable and grown with high structural quality––just like their alloy quasicrystal counterparts.

Quasicrystals are crystals that possess long-range quasiperiodic order without translational periodicity. Since the first report of an icosahedral quasicrystal in an Al–Mn alloy melt (1), quasicrystals with 8-, 10-, 12-, and 18-fold symmetry as well as icosahedral symmetry have been discovered in many alloys (2), carbon allotropes (3, 4), metal oxides (5), and various soft-matter systems including dendrimers (6), block copolymers (7, 8), and colloids (9–12). The apparent structural universality of quasicrystals across these very disparate systems begs the question: Is quasicrystal growth the same across all systems, regardless of length scale?How quasicrystals form is a matter of some debate (13, 14). Two growth models have been proposed: the matching rule model and the error-and-repair model. The matching rule model (15) asserts that quasicrystals form as tiles––i.e., subunits of the quasicrystal pattern comprising clusters of particles in specific arrangements––attach at the growth front to match the existing pattern. Matching rules dictate which attachments are allowed and which are not, and the model imagines that the tiles act as puzzle pieces with precise local fit. Via matching rules, quasicrystals grow “perfectly,” maintaining strict quasiperiodicity at all times, thereby resulting in an ideal (or perfect) tiling. In contrast, the error-and-repair model (16, 17) describes quasicrystal formation as a two-step process. In the first step, tiles or particle clusters quickly attach to the growing quasicrystal in a way that is at least to some degree random. These imperfect attachments eventually produce phason strain, a measure of quasiperiodic disorder over long distances (18, 19). In the second––much slower––step, phason strain is relaxed through local particle rearrangements called phason flips. Ultimately, a quasicrystal with negligible phason strain is possible, so that the end result for both models can be very similar.Recent in situ observations of decagonal (10-fold) quasicrystal growth from Al–Ni–Co melts using transmission electron microscopy (17) and of a self-assembling icosahedral quasicrystal using molecular dynamics simulations from a single-particle species interacting via an isotropic pair potential (20) support the error-and-repair mechanism. While these two systems, one experimental and one computational, are clearly different, in both cases potential energy, rather than entropy, drives quasicrystal growth. Which growth model holds for entropically driven quasicrystal formation (21–23)? There is recent evidence that kinetic crystallization pathways in energy-dominated and entropy-dominated systems are similar (24). Does it follow that soft-matter quasicrystals form via error-and-repair, and thus that high-quality quasicrystals are realizable in entropy-driven soft-matter systems?In this work, we answer this question for the case of the dodecagonal quasicrystal (DQC) in the hard tetrahedron system (25). Because the tetrahedron particles are hard, i.e., interact only via excluded volume, the system we study is governed solely by entropy––that is, the DQC forms from entropy maximization––and thus is a good representation of a soft-matter quasicrystal. We discover from Monte Carlo (MC) simulation runs that phason strain remains small during DQC growth and only weakly relaxes further, resulting in a high-quality DQC with negligible phason strain directly from the melt. We also observe that an approximant structure, a periodic crystal closely resembling the DQC and with inherent linear phason strain (25, 26), can relax to the DQC via continuous phason strain relaxation; that is, the solid–solid transition (27, 28) occurs via a process analogous to the repair step of the error-and-repair model (16, 17).     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

A key requirement for synaptic Reelin signaling in ketamine-mediated behavioral and synaptic action

Ketamine is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist that produces rapid antidepressant action in some patients with treatment-resistant depression. However, recent data suggest that ∼50% of patients with treatment-resistant depression do not respond to ketamine. The factors that contribute to the nonresponsiveness to ketamine’s antidepressant action remain unclear. Recent studies have reported a role for secreted glycoprotein Reelin in regulating pre- and postsynaptic function, which suggests that Reelin may be involved in ketamine’s antidepressant action, although the premise has not been tested. Here, we investigated whether the disruption of Reelin-mediated synaptic signaling alters ketamine-triggered synaptic plasticity and behavioral effects. To this end, we used mouse models with genetic deletion of Reelin or apolipoprotein E receptor 2 (Apoer2), as well as pharmacological inhibition of their downstream effectors, Src family kinases (SFKs) or phosphoinositide 3-kinase. We found that disruption of Reelin, Apoer2, or SFKs blocks ketamine-driven behavioral changes and synaptic plasticity in the hippocampal CA1 region. Although ketamine administration did not affect tyrosine phosphorylation of DAB1, an adaptor protein linked to downstream signaling of Reelin, disruption of Apoer2 or SFKs impaired baseline NMDA receptor–mediated neurotransmission. These results suggest that maintenance of baseline NMDA receptor function by Reelin signaling may be a key permissive factor required for ketamine’s antidepressant effects. Taken together, our results suggest that impairments in Reelin-Apoer2-SFK pathway components may in part underlie nonresponsiveness to ketamine’s antidepressant action.

Major depressive disorder (MDD) is a serious disorder that affects ∼20.6% of the US population and is a leading cause of suicide (1). One crucial problem in treating patients with MDD is an incomplete response rate to medications, where a large fraction of patients do not show a response to primary antidepressant medications (2, 3). Recent clinical findings demonstrate that a subanesthetic dose of ketamine, a noncompetitive N-methyl-d-aspartate receptor (NMDAR) antagonist, produces rapid antidepressant effects within hours in some patients with treatment-resistant depression or MDD (4–6). However, ∼50% of patients with treatment-resistant depression do not respond to ketamine (7), and factors involved in the nonresponsiveness to ketamine remain unclear.The hippocampus is a brain region that has been linked to the pathophysiological changes in MDD. Patients with MDD show a decrease in hippocampal volume and function (8–12). In contrast, MDD patients treated with classic antidepressants have a reversal in hippocampal volume changes along with an improvement in hippocampus-dependent cognitive function (13–15). Previous preclinical studies have shown animal models of depression also exhibit a decrease in hippocampal volume and function (13), and hippocampal synaptic functional enhancement is required to mediate antidepressant responses (16–18). This enhancement of hippocampal function has been suggested to be a key requirement to exert an antidepressant response.Ketamine induces rapid molecular changes that elicit synaptic plasticity in the hippocampus (16, 19–22). Specifically, ketamine rapidly generates synaptic potentiation of field excitatory postsynaptic potentials (fEPSPs) in CA3–CA1 synapses in the hippocampus (ketamine potentiation) by inducing the rapid translation of brain-derived neurotrophic factor (BDNF) and trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) onto the postsynaptic surface (16, 19, 23, 24). Recent studies have shown that if key factors for the antidepressant effects of ketamine, such as BDNF (16, 25, 26) or AMPA receptors (16, 27), are deleted or blocked, the synaptic potentiation in the hippocampus concurrently disappears, suggesting that the synaptic potentiation underlies ketamine’s antidepressant effects (16, 19).Ketamine-mediated potentiation of fEPSPs in CA3–CA1 synapses has been shown to require a block of NMDAR activation by spontaneous glutamate release. Ketamine produces synaptic potentiation in the presence of tetrodotoxin, which blocks sodium channels, and thereby the generation of action potentials, suggesting that blocking NMDARs activated by the spontaneous presynaptic release is key to producing the synaptic potentiation (19, 21, 28, 29). In agreement with this premise, deletion of Vps10p-tail-interactor-1a (Vti1a) and vesicle-associated membrane protein 7 (VAMP7), which are soluble N-ethylmaleimide–sensitive factor attachment protein receptor proteins selectively involved in spontaneous neurotransmitter release (30, 31) in the CA3 hippocampal region, occluded the ketamine potentiation (32). Collectively, these lines of evidence suggest spontaneous glutamate release, and NMDARs are important factors for ketamine potentiation. Thus, it is possible that if these pre- or postsynaptic components are impaired, ketamine may not produce the synaptic potentiation and antidepressant effects.Reelin is a secreted glycoprotein and acts as a neuromodulator in the adult brain by regulating pre- and postsynaptic machinery. Reelin binds to its receptors, apolipoprotein E receptor 2 (Apoer2) and very-low-density lipoprotein receptor (VLDLR) and increases tyrosine phosphorylation in Disabled-1 (DAB1) (33–35). The Reelin pathway regulates pre- or postsynaptic function through its downstream signaling pathways in the adult brain. In presynaptic terminals, the Reelin-Apoer2 pathway activates phosphoinositide 3-kinase (PI3K) and increases Ca²⁺ release from intracellular stores, which in turn mobilizes VAMP7-containing synaptic vesicles and augments spontaneous release (31). At the postsynaptic sites, Reelin’s binding to Apoer2 reciprocally activates DAB1 and Src family kinases (SFKs). Subsequently, the activated SFKs increase tyrosine phosphorylation in NMDAR subunits, GluN2A and GluN2B (34–37), and increase NMDAR open probability (37–39). Since pre- and postsynaptic components regulated by Reelin have been suggested to be important for ketamine potentiation (16, 19–21, 32), it is conceivable that disrupted Reelin signaling may abrogate the antidepressant action and synaptic plasticity of ketamine.To examine this premise, we used genetically modified mice with a deletion of either Reelin or Apoer2 and investigated changes in antidepressant-like behaviors and synaptic potentiation in the CA1 hippocampal region following ketamine treatment. We also used pharmacological inhibitors to examine the effects of signaling molecules downstream of Reelin-Apoer2, specifically SFKs and PI3K, on ketamine-induced behavioral changes and synaptic plasticity. Lastly, we investigated whether the disruption of ketamine’s effects is due to a requirement for the activation of Reelin-dependent signaling or the impairment of NMDAR function by the disruption of Reelin-dependent signaling. Our results suggest that disruption of the Reelin-Apoer2-SFKs pathway depresses NMDAR function and diminishes ketamine’s use-dependent NMDAR antagonism, thereby rendering synapses nonresponsive to ketamine’s action as well as subsequent antidepressant responses. Taken together, these results provide insight into understanding the cellular and molecular mechanisms underlying ketamine’s antidepressant effects.     

Biallelic splicing variants in the nucleolar 60S assembly factor RBM28 cause the ribosomopathy ANE syndrome

Alopecia, neurologic defects, and endocrinopathy (ANE) syndrome is a rare ribosomopathy known to be caused by a p.(Leu351Pro) variant in the essential, conserved, nucleolar large ribosomal subunit (60S) assembly factor RBM28. We report the second family of ANE syndrome to date and a female pediatric ANE syndrome patient. The patient presented with alopecia, craniofacial malformations, hypoplastic pituitary, and hair and skin abnormalities. Unlike the previously reported patients with the p.(Leu351Pro) RBM28 variant, this ANE syndrome patient possesses biallelic precursor messenger RNA (pre-mRNA) splicing variants at the 5′ splice sites of exon 5 (ΔE5) and exon 8 (ΔE8) of RBM28 ({"type":"entrez-nucleotide","attrs":{"text":"NM_018077.2","term_id":"187960108","term_text":"NM_018077.2"}}NM_018077.2:c.[541+1_541+2delinsA]; [946G > T]). In silico analyses and minigene splicing experiments in cells indicate that each splice variant specifically causes skipping of its respective mutant exon. Because the ΔE5 variant results in an in-frame 31 amino acid deletion (p.(Asp150_Lys180del)) in RBM28 while the ΔE8 variant leads to a premature stop codon in exon 9, we predicted that the ΔE5 variant would produce partially functional RBM28 but the ΔE8 variant would not produce functional protein. Using a yeast model, we demonstrate that the ΔE5 variant does indeed lead to reduced overall growth and large subunit ribosomal RNA (rRNA) production and pre-rRNA processing. In contrast, the ΔE8 variant is comparably null, implying that the partially functional ΔE5 RBM28 protein enables survival but precludes correct development. This discovery further defines the underlying molecular pathology of ANE syndrome to include genetic variants that cause aberrant splicing in RBM28 pre-mRNA and highlights the centrality of nucleolar processes in human genetic disease.

Ribosome biogenesis (RB) is the essential cellular process in which the complex macromolecular ribosomal machinery is manufactured and assembled, enabling protein translation (1–4). Both ribosomal RNA (rRNA) and ribosomal protein (RP) components must be correctly synthesized, processed, modified, folded, translocated, and ultimately joined in the cytoplasm to engage in global protein synthesis (1–3). For eukaryotes, four rRNA molecules (1, 5) and about 80 RPs (1, 6, 7) form the core of the mature small (40S) and large (60S) ribosomal subunits. The demand for ribosomes during the cell cycle is immense: in a growing yeast cell, more than 30 ribosomes are synthesized per second (8), while in a growing HeLa cell, this figure balloons to 125 ribosomes per second (9). Over 200 trans-acting assembly factors are necessary to achieve the fast and accurate ribosome assembly required to meet this tremendous cellular translational demand (1).Given that up to 80% of cellular metabolism is devoted to RB (10), it is unsurprising that defects in RB factors are causative of a class of human diseases known as ribosomopathies (1, 11–16). Though not fully understood, tissue-specific defects are the hallmark of ribosomopathies (11, 17). Tissues formed from hematopoietic or neural crest cell lineages are disproportionately affected, resulting in anemia, neutropenia, and leukemia, bone marrow failure diseases including Diamond–Blackfan Anemia (DBA) (18–23) and Shwachman–Diamond syndrome (24–26), craniofacial, dermatological, and neurological diseases including Treacher Collins syndrome (27–29) and postaxial acrofacial dysostosis (30), and alopecia, neurologic defects, and endocrinopathy (ANE) syndrome (31–34).ANE syndrome (OMIM: 612079) (31, 35) is a rare ribosomopathy defined by heterogeneous clinical features of variable severity including alopecia, neurological deformities, intellectual disability, and hormonal deficiencies with pubertal delay. In the only ANE syndrome case report published to date, Nousbeck and coworkers studied five brothers of consanguineous parentage with variable ANE syndrome features, finding that ANE syndrome patient tissue samples had quantifiably fewer ribosomes and qualitatively dysmorphological rough endoplasmic reticula versus healthy control samples (31). All five patients were found to carry a homozygous missense variant (p.(Leu351Pro); L > P) in RBM28 (31), a known essential 60S assembly factor orthologous to yeast Nop4 (36–39). Follow-up studies further defined the clinical extent of endocrinopathy (32) and the biochemical mechanisms of hair and skin defects (33) and of inhibited ribosome biogenesis (34, 40) due to impaired function of RBM28 or its yeast homolog, Nop4. However, due to the rarity of the disease and lack of sufficient animal model studies, further investigation of ANE syndrome has been limited.We report a female pediatric patient in the second family of ANE syndrome to date, unrelated to the family in the original case report (31). The ANE syndrome patient has a clinical presentation consistent with the definition of ANE syndrome but possesses differing genetic variants and molecular pathology. Using in vivo techniques, we demonstrate that the patient’s compound heterozygous splicing variants in RBM28 create one hypomorphic (ΔE5) and one null (ΔE8) allele with respect to overall growth and 60S pre-rRNA processing functions. By elucidating the pathology of an ANE syndrome patient, our results bolster and extend our understanding of this rare ribosomopathy and reinforce the importance of proper nucleolar function in human health and disease.     

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.     

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.