Bifidobacterium alters the gut microbiota and modulates the functional metabolism of T regulatory cells in the context of immune checkpoint blockade

Immune checkpoint-blocking antibodies that attenuate immune tolerance have been used to effectively treat cancer, but they can also trigger severe immune-related adverse events. Previously, we found that Bifidobacterium could mitigate intestinal immunopathology in the context of CTLA-4 blockade in mice. Here we examined the mechanism underlying this process. We found that Bifidobacterium altered the composition of the gut microbiota systematically in a regulatory T cell (Treg)-dependent manner. Moreover, this altered commensal community enhanced both the mitochondrial fitness and the IL-10–mediated suppressive functions of intestinal Tregs, contributing to the amelioration of colitis during immune checkpoint blockade.

Immune checkpoint blockade therapy has become a very successful cancer treatment. The first monoclonal antibody (mAb) approved for clinical use is specific for the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) for melanoma treatment (1). However, the application of immune checkpoint inhibitors (ICIs) can cause various and even fatal autoimmune responses, of which diarrhea and colitis are among the most frequent and severe (2, 3).Components in the gut microbiota have been shown to regulate the host antitumor immune response (4–7), and several studies have implicated the function of the intestinal microbiota in modulating the efficacy of immune checkpoint blockade therapy (5, 8, 9). For example, the presence of Bifidobacterium can stimulate the host immune system to respond to anti–PD-L1 therapy in a CD8⁺ T cell-dependent manner (10).Although these studies have demonstrated a role for the microbiota in antitumor immunity, the underlying events related to checkpoint antibody-induced autoimmunity remain elusive. In the clinic, patients who experienced colitis after ICI treatment harbored gut bacteria compositions that are distinct from those of colitis-free patients (11). A recent study reported the first clinical case in which reconstituting the gut microbiota with fecal microbiota transplantation successfully rescued ICI-associated colitis (9). The baseline of gut microbiota was also shown to be related to that clinical response to ipilimumab, with enrichment of Faecalibacterium consistent with long-term clinical benefit and colitis (12). We previously reported that administration of Bifidobacterium attenuated intestinal inflammation without impairing the antitumor function of CTLA-4 in mice (13). Here we dissect the fundamental principles governing the relationship between the probiotic-induced microbiome optimization and the outcome of CTLA-4 blockade. We demonstrate that Bifidobacterium systematically alters the composition of the gut microbiota, profoundly increasing the other probiotic species, Lactobacillus. This microbiome optimization is dependent on the existence of regulatory T cells (Tregs). Furthermore, we found that both the metabolic and suppressive functions of intestinal Tregs are enhanced by this altered commensal community, contributing to maintaining regional immune homeostasis under the CTLA-4 blockade condition. Taken together, our observations reveal an immunologic principle governing the complex functions of microbiota dynamics, as well as a mechanism for the relay from Bifidobacterium to Lactobacillus in ameliorating immune checkpoint blockade-related colitis.     

CODY enables quantitatively spatiotemporal predictions on in vivo gut microbial variability induced by diet intervention

Microbial variations in the human gut are harbored in temporal and spatial heterogeneity, and quantitative prediction of spatiotemporal dynamic changes in the gut microbiota is imperative for development of tailored microbiome-directed therapeutics treatments, e.g. precision nutrition. Given the high-degree complexity of microbial variations, subject to the dynamic interactions among host, microbial, and environmental factors, identifying how microbiota colonize in the gut represents an important challenge. Here we present COmputing the DYnamics of microbiota (CODY), a multiscale framework that integrates species-level modeling of microbial dynamics and ecosystem-level interactions into a mathematical model that characterizes spatial-specific in vivo microbial residence in the colon as impacted by host physiology. The framework quantifies spatiotemporal resolution of microbial variations on species-level abundance profiles across site-specific colon regions and in feces, independent of a priori knowledge. We demonstrated the effectiveness of CODY using cross-sectional data from two longitudinal metagenomics studies—the microbiota development during early infancy and during short-term diet intervention of obese adults. For each cohort, CODY correctly predicts the microbial variations in response to diet intervention, as validated by available metagenomics and metabolomics data. Model simulations provide insight into the biogeographical heterogeneity among lumen, mucus, and feces, which provides insight into how host physical forces and spatial structure are shaping microbial structure and functionality.

Changes in the human gut microbiome composition are connected with development of numerous diseases, like obesity, type-2 diabetes, and immune dysfunction (1–3). Quantitative understanding and predicting how microbial variations are determined are crucial for designing microbiome-directed therapies that target chronic metabolic diseases (4, 5). However, this remains challenging due to the temporal and spatial heterogeneity along the human gut resulting from a dynamic interplay among host, microbial, and environmental conditions (6, 7). Diet is recognized as a controllable and pivotal environmental factor in shaping longitudinal microbial landscape development (8, 9), such as early childhood colonization (10) and long-term adulthood stabilization (11). While profiling of fecal samples enables a snapshot of consequential changes of the fecal microbiota in response to different stimuli, e.g. dietary changes (12–14), it is still far from describing the intrinsic dynamics of how microbiome colonize in the gut. Recently, the spatial heterogeneity of microbial composition between lumen and mucus has been recognized in mice (15, 16), but similar studies in humans is impossible with current techniques. In addition, measurements of absolute abundance profiles are required to correct the artifacts associated with relative abundance that confound revealing the interplay between microbial variations and health (17). Therefore, methods that enable quantifying the absolute, temporal, and spatial variations of in vivo human gut microbiota are needed to understand how to maintain or restore healthy microbiota.Computational models are widely used to decipher microbial complexity and response to perturbations (18). Most existing models have limited usage as they only address specific elements of the multidimensional interaction mechanisms. For example, similarity-based (19) and rule-mining models (20) describe microbial–microbial interactions without considering temporal dependency. The dynamic Bayesian model enables incorporation of directed interactions and longitudinal dataset (21), while reliance on training dataset and difficulties in model selection render these stochastic models confining to specific statistic condition and predictions are therefore not consistent and generalizable (22). The generalized Lotka–Volterra model (18, 23, 24) represents a step forward to simulate dynamics via formulating microbial growth rate as a lumped term, but adherence to assumptions of pairwise interactions-driven community dynamics and constant environment limits their predictive power. Genome-scale models (GEMs) (25) provide a valuable resource for studying structured microbial metabolism. With GEMs, microbial metabolic capacity, microbe–microbe interactions (26–28), microbial–diet interactions (12), and structural changes of two-species cocultures (29) are characterized using flux balance analysis (FBA). With rare exceptions, FBA requires a priori knowledge of metabolite uptake fluxes distributed among community members, with current limitations on these resources, faces challenges with modeling multispecies communities in a dynamic manner (30). Therefore, in adapting a computational framework that can simulate microbiome dynamics along the human gut, one encounters three challenges: 1) endogenously, the intrinsic dynamics not only emerge from the large number of microbiota components but also through the intricate and dynamic ways they interact (31, 32); 2) exogenously, the microbiota is exposed to a series of host–microbe metabolic axes (33), such as colonic physical forces (34), nascent colonization, and nutrient conditions; and 3) spatial structure of the in vivo microbiota localization plays a significant role impacting 1 and 2 (24).Here, we bridge the current theoretical gap by developing a multiscale framework for COmputing the DYnamics of gut microbiota (CODY), which enables identification and quantification of spatiotemporal-specific variations of gut microbiome absolute and relative abundance profiles, without a prior knowledge of microbiome interactions. We evaluated CODY’s performance by comparing model simulations with longitudinal changes in the microbial composition in fecal samples and in plasma metabolomics of two cohorts: 1) long-term development of the gut microbiome in early infancy and 2) short-term variation patterns of the gut microbiome in obese adults experiencing diet intervention. Comparison of model simulations with experimental data demonstrated predictive strength of the CODY modeling framework and hence lays the foundation for performing design of microbiome-directed therapeutics or of precision nutrition based on CODY simulations. The source code of CODY is freely available together with full documentation at https://github.com/JunGeng-Sysbio-Chalmers/CODY1.0_SourceCode.     

Ammonium transporter expression in sperm of the disease vector Aedes aegypti mosquito influences male fertility

The ammonium transporter (AMT)/methylammonium permease (MEP)/Rhesus glycoprotein (Rh) family of ammonia (NH₃/NH₄⁺) transporters has been identified in organisms from all domains of life. In animals, fundamental roles for AMT and Rh proteins in the specific transport of ammonia across biological membranes to mitigate ammonia toxicity and aid in osmoregulation, acid–base balance, and excretion have been well documented. Here, we observed enriched Amt (AeAmt1) mRNA levels within reproductive organs of the arboviral vector mosquito, Aedes aegypti, prompting us to explore the role of AMTs in reproduction. We show that AeAmt1 is localized to sperm flagella during all stages of spermiogenesis and spermatogenesis in male testes. AeAmt1 expression in sperm flagella persists in spermatozoa that navigate the female reproductive tract following insemination and are stored within the spermathecae, as well as throughout sperm migration along the spermathecal ducts during ovulation to fertilize the descending egg. We demonstrate that RNA interference (RNAi)-mediated AeAmt1 protein knockdown leads to significant reductions (∼40%) of spermatozoa stored in seminal vesicles of males, resulting in decreased egg viability when these males inseminate nonmated females. We suggest that AeAmt1 function in spermatozoa is to protect against ammonia toxicity based on our observations of high NH₄⁺ levels in the densely packed spermathecae of mated females. The presence of AMT proteins, in addition to Rh proteins, across insect taxa may indicate a conserved function for AMTs in sperm viability and reproduction in general.

Ammonium transporters (AMTs), methylammonium permeases (MEPs), and Rhesus glycoproteins (Rh proteins) comprise a protein family with three clades, and homologs from each have been identified in virtually all domains of life (1). AMT proteins were first identified in plants (2) with the simultaneous discovery of MEP proteins in fungi (3), followed by Rh proteins in humans (4). Ammonia (NH₃/NH₄⁺) is vital for growth in plants and microorganisms and is retained in some animals for use as an osmolyte (5, 6), for buoyancy (7, 8), and for those lacking sufficient dietary nitrogen (9). In the majority of animals, however, ammonia is the toxic by-product of amino acid and nucleic acid metabolism and, accordingly, requires efficient mechanisms for its regulation, transport, and excretion (10–13). AMT, MEP, and Rh proteins are responsible for the selective movement of ammonia (NH₃) or ammonium (NH₄⁺) across biological membranes, a process that all organisms require. Unlike their vertebrate, bacterial, and fungal counterparts which function as putative NH₃ gas channels (14–18), a myriad of evidence suggests that plant AMT proteins and closely related members in some animals are functionally distinct and facilitate electrogenic ammonium (NH₄⁺) transport (17, 19–22). In contrast to vertebrates which only possess Rh proteins (23), many invertebrates are unique in that they express both AMT and Rh proteins, sometimes in the same cell (24–28). Among insects, the presence of both AMT and Rh proteins has been described in Drosophila melanogaster (29, 30) and mosquitoes that vector disease-causing pathogens, Anopheles gambiae (22, 31) and Aedes aegypti (32, 33). It is unclear whether, in these instances, AMT and Rh proteins can functionally substitute for one another, but in the anal papillae of A. aegypti larvae, knockdown of either Amt or Rh proteins causes decreases in ammonia transport, suggesting that they do not (32–34). To date, studies on ammonia transporter (AMT and Rh) function in insects have focused on ammonia sensing and tasting in sensory structures (22, 30, 31, 35), ammonia detoxification and acid–base balance in muscle, digestive, and excretory organs (15, 36), and ammonia excretion in a variety of organs involved in ion and water homeostasis (9, 24, 32–34).A. aegypti is the primary vector for the transmission of the human arboviral diseases Zika, yellow fever, chikungunya, and dengue virus, which are of global health concern due to rapid increases in the geographical distribution of this species, presently at its highest ever (37, 38). In light of the well-documented evolution of insecticide resistance in mosquitoes (39–42), more recent methods to control disease transmission such as the sterile insect technique (43), transinfection and sterilization of mosquitoes with the bacterium Wolbachia (44), and targeted genome editing rendering adult males sterile (45) have proven effective. These methods take advantage of various aspects of mosquito reproductive biology; however, an understanding of male reproductive biology and the male contributions to female reproductive processes is still in its infancy (46). Here, we describe the expression of an A. aegypti ammonium transporter (AeAmt1) in the sperm during all stages of spermatogenesis, spermiogenesis, and egg fertilization, which is critical for fertility.     

Microbiome reduction and endosymbiont gain from a switch in sea urchin life history

Animal gastrointestinal tracts harbor a microbiome that is integral to host function, yet species from diverse phyla have evolved a reduced digestive system or lost it completely. Whether such changes are associated with alterations in the diversity and/or abundance of the microbiome remains an untested hypothesis in evolutionary symbiosis. Here, using the life history transition from planktotrophy (feeding) to lecithotrophy (nonfeeding) in the sea urchin Heliocidaris, we demonstrate that the lack of a functional gut corresponds with a reduction in microbial community diversity and abundance as well as the association with a diet-specific microbiome. We also determine that the lecithotroph vertically transmits a Rickettsiales that may complement host nutrition through amino acid biosynthesis and influence host reproduction. Our results indicate that the evolutionary loss of a functional gut correlates with a reduction in the microbiome and the association with an endosymbiont. Symbiotic transitions can therefore accompany life history transitions in the evolution of developmental strategies.

Animal gastrointestinal tracts contain microbial communities that are integral to host metabolism, immunity, and development (1, 2). Symbioses between animals and their gut microbiome have deep evolutionary origins (1, 2), often exhibit phylosymbiosis (3), and can serve as a physiological buffer to heterogeneous environments (2). Despite the necessity of the gastrointestinal tract and benefits of the gut microbiome (3), species in various phyla have lost a functional digestive system (4, 5). Loss of a functional gut should, in theory, cascade into a reduction in microbial diversity and the loss of diet-induced shifts in microbiome composition. These nutritional shifts may then provide a niche for functionally important endosymbionts, such as the chemoautotrophic bacteria commonly associated with gutless invertebrates (6, 7).Major life history transitions are driven by tradeoffs in reproduction and development that, in turn, impact fitness (8). These tradeoffs are particularly evident in benthic marine invertebrates whose developmental stages broadly group into two alternative nutritional strategies (4, 9). The first—planktotrophy—typically includes the production of a high number of small, energy-poor eggs that develop into larvae with feeding structures used to collect and process exogenous resources required to reach metamorphic competency (4, 9). The second—lecithotrophy—involves the production of fewer large, energy-rich eggs and nonfeeding larvae that undergo metamorphosis without the requirement of external nutrients through feeding (4, 9). Life history transitions between these developmental modes have occurred in several major animal lineages, with rapid evolutionary shifts from planktotrophy to lecithotrophy being well documented in echinoderms (4, 5, 10–13). It is thought that an increase in the eggs energetic content relaxes the selective pressure maintaining the feeding structures (e.g., the larval arms and a functional gastrointestinal tract) and that development to metamorphosis is accelerated once these are lost (5).One of the most comprehensively studied systems for life history transitions among marine invertebrates involves species in the sea urchin genus Heliocidaris. A speciation event ∼5 Mya resulted in two sister species with alternative life history strategies: Heliocidaris tuberculata is planktotrophic while Heliocidaris erythrogramma is lecithotrophic (14). Typical of planktotrophs, H. tuberculata develops from small eggs into feeding larvae that exhibit morphological plasticity in response to food limitation (15), which is correlated with compositional shifts in the microbiome (16, 17). H. erythrogramma, on the other hand, develops from eggs ∼53× to 86× the volume of H. tuberculata (18), lacks the morphological structures required for feeding, and has a reduced, nonfunctional digestive tract (11). This life history switch and heterochronic shift in development (11) corresponds with a rewiring of the gene regulatory network (19), reorganization of cell fates (20), and modification to gametogenesis (21).Here, we compare the bacterial communities of these Heliocidaris species and test two hypotheses. First, we test whether the loss of gut function coincides with a reduction in microbial symbiont diversity, and second, by simulating the natural range in food availability, we also test that the loss in gut function coincides with a loss in diet-related shifts in the microbiome. We report major reductions in microbiome diversity and abundance as well as the absence of bacterial communities correlated with food availability for the lecithotrophic H. erythrogramma. Moreover, we find that this species vertically transmits a Rickettsiales that encodes pathways for the biosynthesis of essential amino acids, proteins with pivotal roles in host reproduction, and enzymes to metabolize diacylglycerol ethers, the major lipid group responsible for the increase in egg size in H. erythrogramma and that is used to fuel growth and development (18, 22).     

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.     

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.     

A machine-learning approach to map landscape connectivity in Aedes aegypti with genetic and environmental data

Mapping landscape connectivity is important for controlling invasive species and disease vectors. Current landscape genetics methods are often constrained by the subjectivity of creating resistance surfaces and the difficulty of working with interacting and correlated environmental variables. To overcome these constraints, we combine the advantages of a machine-learning framework and an iterative optimization process to develop a method for integrating genetic and environmental (e.g., climate, land cover, human infrastructure) data. We validate and demonstrate this method for the Aedes aegypti mosquito, an invasive species and the primary vector of dengue, yellow fever, chikungunya, and Zika. We test two contrasting metrics to approximate genetic distance and find Cavalli-Sforza–Edwards distance (CSE) performs better than linearized F_ST. The correlation (R) between the model’s predicted genetic distance and actual distance is 0.83. We produce a map of genetic connectivity for Ae. aegypti’s range in North America and discuss which environmental and anthropogenic variables are most important for predicting gene flow, especially in the context of vector control.

Landscape genetics—explicitly quantifying the effects of a heterogenous landscape on gene flow—is an important tool for both conservation biology and the control of invasive species and disease vectors including the “yellow fever mosquito” (Aedes aegypti) (1, 2). We demonstrate that current limitations in landscape genetics can be addressed with a machine-learning approach integrated into an iterative optimization process. Isolation by distance (IBD) is a classical model in population genetics that assumes dispersal is limited in proportion to geographic distance, resulting in increasing genetic differentiation with increasing geographic distance between populations (3–5). Although this pattern is commonly seen in nature, factors such as history and dispersal limitations caused by the environment (i.e., “isolation by resistance”) (6) can produce deviations from IBD. Landscape resistance (alias friction) and its inverse, connectivity, determine how organisms move through a landscape (7). Modeling landscape connectivity can be used to identify the environmental variables that affect the organisms’ gene flow and genetic structure; predict how climate and land use change will affect their gene flow and distribution in the future; and inform conservation, vector control, and other management decisions (1, 8–13). Our goals are to use environmental data (the predictors) to build a model of genetic connectivity (the observed data) that improves on IBD and to identify environmental drivers of gene flow patterns.We implement a machine-learning approach that offers a number of advantages over classical methods in landscape genetics: The machine-learning approach is more objective, it allows the inclusion of correlated variables, and it is able to account for different shapes and magnitudes of correlations between predictor and response variables at different locations in the landscape (14–17). In comparison, a common approach in landscape genetics called resistance surface mapping involves the subjective process of creating resistance surfaces for environmental variables, in which each pixel represents a hypothesized resistance to the organism’s movement often based on expert opinion (6, 18). Effective landscape distances through the resistance surfaces can be found with least cost path or circuit theory analysis (19) and then analyzed for associations with genetic distance (20).One option to circumvent the subjectivity of creating resistance surfaces is to model genetic connectivity directly from environmental data. Bouyer et al. (7) took this approach and used a maximum-likelihood method to integrate genetic data and environmental data to map landscape resistance in tsetse flies. Additionally, they introduced an iterative optimization approach in which each subsequent iteration used least cost path lines through the previously predicted resistance surface—an improvement over modeling organism movement as straight lines (16, 17). While this presented a major advance, the maximum-likelihood methodology requires exclusion of correlated data, establishing the relationship between environmental variables and genetic distance before building the model, and transforming or discretizing nonlinear relationships. Additionally, this approach assumes one relationship between each environmental variable and the genetic data across the whole landscape. To build on previous advances while overcoming some of their limitations, we combine iterative optimization with a machine-learning method called random forest (RF).RF is a nonlinear classification and regression tree analysis that can handle many inputs, including redundant or irrelevant variables, as well as continuous and categorical data types (14, 15). RF creates many internal training/testing subdatasets and aggregates the predictors, resulting in stable and consistent results that generally do not overfit the data and can be evaluated through validation processes (14). It is easier to tune and less likely to overfit noisy data than another machine-learning method we considered, gradient boosting (21). Additionally, RF has been successfully incorporated into ecological studies (22) and a small number of landscape genetics studies (16, 17, 23). These studies considered only the environmental predictor values at the genetic collection sites (23) or along straight lines between each pair of sites (16, 17), in contrast to the least cost path analysis we implement here (7).We demonstrate the efficacy of our method to map landscape connectivity for an important disease vector. Ae. aegypti is highly invasive and the primary vector of yellow fever, Zika, dengue, and chikungunya. Except for yellow fever, there are no reliable, widely used vaccines for these diseases, so vector control is essential. Ae. aegypti originated in Africa and is now found throughout the tropics and increasingly in temperate regions (24–26). The species is temperature constrained, preferring warm, humid areas close to humans (the females’ preferred source for bloodmeals outside their native African range) (27). In the United States, it has a patchy distribution throughout southern states, especially Texas, Florida, and California (28). Although Ae. aegypti can disperse >1 km, its usual lifetime dispersal is only around 200 m (29–32). Passive “hitchhiking” via human transportation networks is responsible for long-distance invasions and worldwide spread of Ae. aegypti and its close relative (33–35). Climate change is also expanding the range of Aedes species, which could expose nearly 1 billion additional people to diseases carried by these mosquitoes for the first time (26).Although IBD is common in nature and a helpful null model in landscape genetics (20), geographic distance is often an inadequate sole predictor of genetic distance (as in the case of our dataset; SI Appendix, Fig. S1). Therefore, a more complex model is needed to explain and predict genetic distance and corresponding landscape connectivity. In this paper we introduce an iterative machine-learning approach to integrate environmental predictors and genetic observation data and apply it to map landscape connectivity for the Ae. aegypti mosquito in North America. We also find and examine the most important variables for building the connectivity model and provide validation of our proposed method.     

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.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor⁺ (LepR⁺) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

The maintenance and repair of the skeleton require the generation of new bone cells throughout adult life. Osteoblasts are relatively short-lived cells that are constantly regenerated, partly by skeletal stem cells within the bone marrow (1). The main source of new osteoblasts in adult bone marrow is leptin receptor-expressing (LepR⁺) stromal cells (2–4). These cells include the multipotent skeletal stem cells that give rise to the fibroblast colony-forming cells (CFU-Fs) in the bone marrow (2), as well as restricted osteogenic progenitors (5) and adipocyte progenitors (6–8). LepR⁺ cells are a major source of osteoblasts for fracture repair (2) and growth factors for hematopoietic stem cell maintenance (9–11).One growth factor synthesized by LepR⁺ cells, as well as osteoblasts and osteocytes, is osteolectin/Clec11a, a secreted glycoprotein of the C-type lectin domain superfamily (5, 12, 13). Osteolectin is an osteogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR⁺ cells into osteoblasts. Osteolectin acts by binding to integrin α11β1, which is selectively expressed by LepR⁺ cells and osteoblasts, activating the Wnt pathway (12). Deficiency for either Osteolectin or Itga11 (the gene that encodes integrin α11) reduces osteogenesis during adulthood and causes early-onset osteoporosis in mice (12, 13). Recombinant osteolectin promotes osteogenic differentiation by bone marrow stromal cells in culture and daily injection of mice with osteolectin systemically promotes bone formation.Osteoporosis is a progressive condition characterized by reduced bone mass and increased fracture risk (14). Several factors contribute to osteoporosis development, including aging, estrogen insufficiency, mechanical unloading, and prolonged glucocorticoid use (14). Existing therapies include antiresorptive agents that slow bone loss, such as bisphosphonates (15, 16) and estrogens (17), and anabolic agents that increase bone formation, such as parathyroid hormone (PTH) (18), PTH-related protein (19), and sclerostin inhibitor (SOSTi) (20). While these therapies increase bone mass and reduce fracture risk, they are not a cure.PTH promotes both anabolic and catabolic bone remodeling (21–24). PTH is synthesized by the parathyroid gland and regulates serum calcium levels, partly by regulating bone formation and bone resorption (23–25). PTH1R is a PTH receptor (26, 27) that is strongly expressed by LepR⁺ bone marrow stromal cells (8, 28–30). Recombinant human PTH (Teriparatide; amino acids 1 to 34) and synthetic PTH-related protein (Abaloparatide) are approved by the US Food and Drug Administration (FDA) for the treatment of osteoporosis (19, 31). Daily (intermittent) administration of PTH increases bone mass by promoting the differentiation of osteoblast progenitors, inhibiting osteoblast and osteocyte apoptosis, and reducing sclerostin levels (32–35). PTH promotes osteoblast differentiation by activating Wnt and BMP signaling in bone marrow stromal cells (28, 36, 37), although the mechanisms by which it regulates Wnt pathway activation are complex and uncertain (38).Sclerostin is a secreted glycoprotein that inhibits Wnt pathway activation by binding to LRP5/6, a widely expressed Wnt receptor (7, 8), reducing bone formation (39, 40). Sclerostin is secreted by osteocytes (8, 41), negatively regulating bone formation by inhibiting the differentiation of osteoblasts (41, 42). SOSTi (Romosozumab) is a humanized monoclonal antibody that binds sclerostin, preventing binding to LRP5/6 and increasing Wnt pathway activation and bone formation (43). It is FDA-approved for the treatment of osteoporosis (20, 44) and has activity in rodents in addition to humans (45, 46).The discovery that osteolectin is a bone-forming growth factor raises the question of whether it mediates the effects of PTH or SOSTi on osteogenesis.     

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.     

From the Cover: Suppression of female fertility in Aedes aegypti with a CRISPR-targeted male-sterile mutation

Aedes aegypti spread devastating viruses such as dengue, which causes disease among 100 to 400 million people annually. A potential approach to control mosquito disease vectors is the sterile insect technique (SIT). The strategy involves repeated release of large numbers of sterile males, which reduces insect populations because the sterile males mate and thereby suppress the fertility of females that would otherwise mate with fertile males. While SIT has been successful in suppressing certain agricultural pests, it has been less effective in depressing populations of Ae. aegypti. This limitation is in part because of the fitness effects resulting from mutagenizing the mosquitoes nonspecifically. Here, we introduced and characterized the impact on female fertility of an Ae. aegypti mutation that disrupts a gene that is specifically expressed in testes. We used CRISPR/Cas9 to generate a null mutation in the Ae. aegypti β2-tubulin (B2t) gene, which eliminates male fertility. When we allowed wild-type females to first mate with B2t mutant males, most of the females did not produce progeny even after being subsequently exposed to wild-type males. We also introduced B2t mutant and wild-type males simultaneously with wild-type females and found that a larger number of B2t mutant males relative to the wild-type males was effective in significantly suppressing female fertility. These results raise the possibility of employing B2t sterile males to improve the efficacy of SIT in suppressing populations of Ae. aegypti through repeated releases and thereby reduce the transmission of viruses by these invasive mosquitoes.

Aedes aegypti transmit dengue and Zika and other viral pathogens that cause disease among many tens of millions of people each year (1). Moreover, the incidence of diseases spread by this invasive mosquito continues to be on the rise (2). Only females spread disease, and this occurs because they take a blood meal to obtain nutrients needed for egg production. One potential approach for controlling Ae. aegypti is the sterile insect technique (SIT) (3, 4). SIT involves inundating a local population with large excesses of sterile males. Sterile males are thought to cause sterility in females because the initial mating prevents successful insemination by a wild-type male (1, 3). SIT involves repeated release of large numbers of males in a defined geographical region until the number of females falls below a critical threshold necessary for sustaining the population at a high level.To create sterile males for implementation of SIT, male mosquitoes are exposed to high doses of chemicals or radiation (5). These approaches suffer from the limitation that they do not specifically target male fertility genes (5). Rather, they induce mutations in many genes. This leads to a fitness deficit, reducing the overall robustness of the mosquitoes and the ability of the sterilized males to compete with males in the wild.In addition to classical SIT, many other approaches are being pursued including the release of fertile male insects with a dominant lethal mutation that kills females (6, 7). A very exciting new technology is the use of CRISPR/Cas9 to create gene drives that suppress mosquito populations (8–12). Despite the great promise of these strategies, the release of fertile transgenic mosquitoes creates public concern due to the potential for unintended consequences resulting from these insects lingering in the environment (13).In addition to the classical SIT approach, sterility can also be induced by release of male mosquitoes infected with endosymbiotic strains of Wolbachia (14). The bacteria are present in the testis and ovaries and can disrupt reproductive capacity through multiple mechanisms including cytoplasmic incompatibility (incompatible insect technique; IIT), which occurs following mating between an infected male and a female that is not infected with the same strain of Wolbachia (15). While IIT is a very exciting approach with examples of success (16–18), Wolbachia could spread through an indigenous population, thereby interfering with its efficacy, or get introduced into areas that are not intended to be targeted.The classical SIT approach that involves release of sterile males has multiple advantages that motivate continuing interest in improving this strategy. It has been highly effective in controlling insect pests over the course of 65 y (19, 20), the strategy is environmentally sound (3), and SIT does not suffer from a high level of concern about introduction of transgenes into native insect populations since the released males are sterile. Nevertheless, due to the adverse effects of nonspecific chemical- or radiation-induced sterility, the efficacy of SIT in suppressing mosquito vectors is limited (5). Potentially, SIT could be improved by an alternative, genetic approach using sterile males with a targeted mutation in a single gene that is specifically required for male fertility. However, no male fertility gene has been defined experimentally in mosquitoes.To identify an Ae. aegypti gene likely to cause male sterility, we considered Drosophila male fertility genes that are conserved in these mosquitoes. The Ae. aegypti β2 tubulin (B2t) gene was a prime candidate for eliminating male fertility, since Drosophila B2t is required for male fertility, is specifically expressed in Drosophila sperm (21–23), and the Drosophila and Ae. aegypti proteins are 96% identical. Moreover, the Ae. aegypti B2t gene appears to be expressed specifically in testes (24). In this study, we used CRISPR/Cas9 to knock out the B2t gene. The Ae. aegypti B2t mutant males were sterile and were as robust as wild type in terms of all parameters measured, except for the sterility. Of primary importance, the B2t mutant males were effective in suppressing female fertility. These data indicate that mutation of B2t provides a targeted genetic strategy for improving SIT.     

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.     

Spatial distribution of LTi-like cells in intestinal mucosa regulates type 3 innate immunity

Lymphoid tissue inducer (LTi)-like cells are tissue resident innate lymphocytes that rapidly secrete cytokines that promote gut epithelial integrity and protect against extracellular bacterial infections.Here, we report that the retention of LTi-like cells in conventional solitary intestinal lymphoid tissue (SILT) is essential for controlling LTi-like cell function and is maintained by expression of the chemokine receptor CXCR5. Deletion of Cxcr5 functionally unleashed LTi-like cells in a cell intrinsic manner, leading to uncontrolled IL-17 and IL-22 production. The elevated production of IL-22 in Cxcr5-deficient mice improved gut barrier integrity and protected mice during infection with the opportunistic pathogen Clostridium difficile. Interestingly, Cxcr5^−/− mice developed LTi-like cell aggregates that were displaced from their typical niche at the intestinal crypt, and LTi-like cell hyperresponsiveness was associated with the local formation of this unconventional SILT. Thus, LTi-like cell positioning within mucosa controls their activity via niche-specific signals that temper cytokine production during homeostasis.

Lymphoid tissue inducer (LTi)-like cells belong to a family of tissue resident innate lymphocytes that lack rearranged antigen-specific receptors and act as a first line of defense at barrier tissues. LTi-like cells, along with other group 3 innate lymphoid cells (ILC3), maintain intestinal homeostasis by producing the cytokines IL-22 and IL-17A, which promote gut epithelial cell proliferation, anti-microbial peptide production, and tight junction protein abundance (1, 2). The conditioning of epithelial cells by these cytokines contributes to balanced interactions between the host and commensal microbiota under steady-state conditions, and LTi-like cell-derived IL-22 promotes barrier integrity and protective immunity during infection with the enteric pathogenic bacteria (3).In addition to providing effector functions, LTi-like cells and their fetal LTi counterparts are required for early steps in lymphoid tissue development. Fetal LTi induce lymph node and Peyer’s patch development during gestation by activating lymphoid tissue organizer cells at primordial lymphoid organs with lymphotoxin (LT)-α1β2 (4–6). Similarly, LTi-like cells are required for the postnatal development of cryptopatches, small lymphoid aggregates in the intestine that have the potential to mature into isolated lymphoid follicles (ILF) in response to signals from microbes (7, 8). In line with their roles in lymphoid tissue organogenesis and maturation, LTi-like cells in adult mouse intestines preferentially localize in solitary intestinal lymphoid tissue (SILT). The microenvironments of these highly specialized niches are expected to support and regulate LTi-like cells; however, their impact on LTi-like cell behavior has not been fully explored.LTi-like cells express multiple G protein–coupled receptors that facilitate their migration in tissue (9–12). Among these, CXCR5 has a predominant role in the migration of LTi to developing lymphoid structures, with Cxcr5^−/− mice exhibiting defects in lymph node and Peyer’s patch development (13). Mice deficient in CXCR5 or its ligand CXCL13 also have delayed cryptopatch development and fail to convert cryptopatches to mature ILF because of impaired recruitment of B cells to these structures (14–16). Dendritic cells (DCs) have been shown to be a local source of CXCL13 in SILT (16) and thus likely retain B cells and LTi-like cells at these structures under homeostatic conditions via the CXCL13–CXCR5 signaling axis. The retention of LTi-like cells in SILT is expected to bring these cells in close proximity to activating and inhibitory signals provided by specialized myeloid cells, neurons that express the vasoactive intestinal peptide (VIP), and lymphocyte populations localized at these sites (17–20). However, the impact of CXCR5 on functions of LTi-like cells beyond those associated with lymphoid tissue maintenance and development remains unknown.In the current study, we show that CXCR5 expression regulates LTi-like cell function. Deletion of Cxcr5 led to increased numbers of LTi-like cells in the small intestine (SI) and enhanced their ability to produce IL-17A and IL-22. Cxcr5 regulated LTi-like cells via a cell-intrinsic mechanism that did not involve direct suppression by CXCL13. Heightened LTi-like cell activity in Cxcr5-deficient mice was associated with the development of abnormal LTi-like cell aggregates in the SI that were localized in villus lamina propria instead of at the intestinal crypt base. Importantly, augmented production of IL-22 in Cxcr5^−/− mice was protective during acute infection with the opportunistic pathogen Clostridium difficile. These data reveal that CXCR5-dependent migration can control innate type 3 immunity by altering the niche of LTi-like cells in intestinal lamina propria.