Iron robbery by intracellular pathogen via bacterial effector–induced ferritinophagy

Iron is essential for survival and proliferation of Ehrlichia chaffeensis, an obligatory intracellular bacterium that causes an emerging zoonosis, human monocytic ehrlichiosis. However, how Ehrlichia acquires iron in the host cells is poorly understood. Here, we found that native and recombinant (cloned into the Ehrlichia genome) Ehrlichia translocated factor-3 (Etf-3), a previously predicted effector of the Ehrlichia type IV secretion system (T4SS), is secreted into the host cell cytoplasm. Secreted Etf-3 directly bound ferritin light chain with high affinity and induced ferritinophagy by recruiting NCOA4, a cargo receptor that mediates ferritinophagy, a selective form of autophagy, and LC3, an autophagosome biogenesis protein. Etf-3−induced ferritinophagy caused ferritin degradation and significantly increased the labile cellular iron pool, which feeds Ehrlichia. Indeed, an increase in cellular ferritin by ferric ammonium citrate or overexpression of Etf-3 or NCOA4 enhanced Ehrlichia proliferation, whereas knockdown of Etf-3 in Ehrlichia via transfection with a plasmid encoding an Etf-3 antisense peptide nucleic acid inhibited Ehrlichia proliferation. Excessive ferritinophagy induces the generation of toxic reactive oxygen species (ROS), which could presumably kill both Ehrlichia and host cells. However, during Ehrlichia proliferation, we observed concomitant up-regulation of Ehrlichia Fe-superoxide dismutase, which is an integral component of Ehrlichia T4SS operon, and increased mitochondrial Mn-superoxide dismutase by cosecreted T4SS effector Etf-1. Consequently, despite enhanced ferritinophagy, cellular ROS levels were reduced in Ehrlichia-infected cells compared with uninfected cells. Thus, Ehrlichia safely robs host cell iron sequestered in ferritin. Etf-3 is a unique example of a bacterial protein that induces ferritinophagy to facilitate pathogen iron capture.

Ehrlichia spp., rickettsial obligatory intracellular bacteria cause tick-borne infectious diseases, which are greatly rising in worldwide prevalence (1, 2). Ehrlichia chaffeensis causes human monocytic ehrlichiosis, a severe flu-like illness accompanied by hematologic abnormalities and hepatitis, which can be fatal (2 to 5% mortality) (3–6). In humans, E. chaffeensis infects monocytes and macrophages and concocts unique membrane-bound compartments (inclusions). The inclusions have early endosome-like characteristics, including the presence of transferrin (Tf), transferrin receptor (TfR), and vacuolar-type H⁺-ATPase as well as the small GTPase RAB5 and its effectors, but the inclusions lack late endosomal or lysosomal markers or NADPH oxidase (7–9). Within the inclusions, Ehrlichia acquires all nutrients, including iron, for its reproduction to yield numerous mature infectious forms.Iron serves as a cofactor in many processes of bacteria and eukaryotes, including electron transfer, energy metabolism, oxygen transport, oxygen sensing, and DNA synthesis and repair (10). Ehrlichia is an obligate aerobe and is absolutely dependent on host iron for ATP synthesis via the electron transport chain, because its glycolytic pathway is incomplete and it lacks ATP−ADP translocase, unlike Rickettsia and Chlamydia (11). E. chaffeensis lacks the siderophore biosynthesis pathway and Fe³⁺ uptake regulator (11). Nonetheless, Ehrlichia acquires iron from the host cell labile cellular iron (LCI) pool, and pretreating human monocytes with the membrane-permeable iron chelator deferoxamine blocks E. chaffeensis infection (12). Ehrlichia enhances host cell iron uptake via up-regulating TfR messenger RNA (mRNA) (13) and acquires iron from the holoTf, as E. chaffeensis endosomes intersect with TfR-recycling endosomes and are slightly acidic—enough to release iron from holoTf (7). In fact, treatment of macrophages with interferon-γ down-regulates TfR mRNA and almost completely inhibits Ehrlichia infection, and addition of holoTf abrogates this inhibition (12). However, TfR mRNA levels return to basal level after 24 h postinfection when bacterial exponential growth begins (13), and treatment with interferon-γ can no longer inhibit infection at this point (12), suggesting that alternative or additional iron acquisition mechanisms exist to support exponential intracellular growth of Ehrlichia.The bacterial type IV secretion system (T4SS) translocates bacterial proteins and nucleoprotein complexes (called “effectors,” as they bring about responses) from bacteria to eukaryotic cells (14). Rickettsial organisms including E. chaffeensis have T4SS, sometimes referred to as T4aSS, similar to the virB/virD system of Agrobacterium tumefaciens (15, 16). The dot/icm system of Legionella pneumophila, sometimes referred to as T4bSS, secretes ∼300 effectors with redundant functions; hence, each effector can be knocked out, but the mutant lacks a phenotype (17). In contrast, the total number of T4aSS effectors is much lower [for example, fewer than six effectors exist in A. tumefaciens (15)], but each effector has a crucial role in infection/disease. To date, only a handful of T4SS effectors have been identified for rickettsial organisms, and even fewer have been functionally characterized (18–21). VirD4 is a well-established coupling protein involved in escorting translocated DNA and proteins in A. tumefaciens (15). By a bacterial two-hybrid screen using E. chaffeensis VirD4 as bait, we previously identified three Ehrlichia proteins that directly bind to Ehrlichia VirD4: ECH0825 (Ehrlichial translocated factor-1, Etf-1), ECH0261 (Etf-2), and ECH0767 (Etf-3) (22). All three were formerly annotated as hypothetical proteins, as they lack sequence similarity to previously known proteins or protein domains or motifs. Etf-1 has key roles in Ehrlichia infection of human cells by blocking host cell apoptosis and inducing RAB5-regulated autophagy for nutrient (amino acids) acquisition (9, 22, 23). Etf-2 is a unique RABGAP5 structural mimic that lacks GTPase-activating protein activity (24). Etf-2 directly binds RAB5-GTP on the Ehrlichia inclusion membrane and impedes the fusion of Ehrlichia-containing early endosomes with lysosomes (24). Whether Etf-3 is secreted or has any biological function is unknown. In the present study, we discovered that Etf-3 is a true T4SS effector that is secreted, binds tightly to ferritin, and induces ferritinophagy to provide free Fe²⁺ for intracellular Ehrlichia.     

A conserved Ctp1/CtIP C-terminal peptide stimulates Mre11 endonuclease activity

The Mre11-Rad50-Nbs1 complex (MRN) is important for repairing DNA double-strand breaks (DSBs) by homologous recombination (HR). The endonuclease activity of MRN is critical for resecting 5′-ended DNA strands at DSB ends, producing 3′-ended single-strand DNA, a prerequisite for HR. This endonuclease activity is stimulated by Ctp1, the Schizosaccharomyces pombe homolog of human CtIP. Here, with purified proteins, we show that Ctp1 phosphorylation stimulates MRN endonuclease activity by inducing the association of Ctp1 with Nbs1. The highly conserved extreme C terminus of Ctp1 is indispensable for MRN activation. Importantly, a polypeptide composed of the conserved 15 amino acids at the C terminus of Ctp1 (CT15) is sufficient to stimulate Mre11 endonuclease activity. Furthermore, the CT15 equivalent from CtIP can stimulate human MRE11 endonuclease activity, arguing for the generality of this stimulatory mechanism. Thus, we propose that Nbs1-mediated recruitment of CT15 plays a pivotal role in the activation of the Mre11 endonuclease by Ctp1/CtIP.

DNA double-strand breaks (DSBs) are potentially lethal lesions that threaten genomic integrity and cell viability. DSBs can occur spontaneously as a result of faulty DNA metabolism or by exposure to genotoxins. In eukaryotes, these DSBs have “dirty ends” that lack ligatable 3ʹ-hydroxyl/5ʹ-phosphate groups and are often firmly attached to proteins such as the Ku70-80 heterodimer and topoisomerases (1, 2). During meiosis, the topoisomerase-like Spo11 protein generates DSBs and remains covalently attached to the 5ʹ DNA ends (3). To enable further processing, these DSB ends must be converted to “clean” ends with 3ʹ-hydroxyl/5ʹ-phosphate groups properly exposed. This step is achieved by endonucleolytic cleavage, or clipping, by Mre11 (4–7).In mammals, the MRE11, RAD50, and NBS1 complex (Mre11-Rad50-Nbs1 [MRN]), together with CtIP, is involved in the clipping reaction. MRE11 is the nuclease subunit that has both endonuclease and 3′-to-5′ exonuclease activities, but only the former is essential for clipping (4, 8–12). RAD50, a member of the Structural Maintenance of Chromosomes protein family, binds to MRE11 to form an (MRE11)₂-(RAD50)₂ ring structure (MR complex) (13–15). NBS1 binds to the MR complex via MRE11 to form the MRN complex (16). Homologs of CtIP include Ctp1 in Schizosaccharomyces pombe and Sae2 in Saccharomyces cerevisiae (17–19). Upon phosphorylation, these proteins physically interact with their cognate MRN complex via the N-terminal forkhead-associated domain of NBS1, leading to activation of the MRE11 endonucleolytic clipping activity (20–24). However, the mechanistic details underlying this activation have not yet been determined.Through biochemical reconstitution using fission yeast proteins, we made three key findings regarding how Ctp1 activates MRN. First, MRN activation is mediated by Ctp1 phosphorylation, which promotes the direct association of Ctp1 with the Nbs1 subunit of MRN. Second, the highly conserved extreme C terminus of Ctp1 retains the ability to promote the endonuclease activity of MRN. Strikingly, a synthetic polypeptide comprising the 15 amino acids from the extreme C terminus of Ctp1 was sufficient for the full activation of MRN. Third, we verified the evolutionary significance of these findings by demonstrating that the conserved C-terminal polypeptide of CtIP can also stimulate the endonuclease activity of human MRN. Together, our results strongly suggest that the Ctp1-promoted MRN activation mechanism consists of at least two fundamentally separable elements: phosphorylation-induced Ctp1-MRN association and activation of MRN by the C-terminal peptide of Ctp1. Thus, recruitment of the Ctp1 C terminus to MRN is likely pivotal in this activation mechanism.     

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.     

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.     

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.     

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.     

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.     

Wild-type α-synuclein inherits the structure and exacerbated neuropathology of E46K mutant fibril strain by cross-seeding

Heterozygous point mutations of α-synuclein (α-syn) have been linked to the early onset and rapid progression of familial Parkinson’s diseases (fPD). However, the interplay between hereditary mutant and wild-type (WT) α-syn and its role in the exacerbated pathology of α-syn in fPD progression are poorly understood. Here, we find that WT mice inoculated with the human E46K mutant α-syn fibril (hE46K) strain develop early-onset motor deficit and morphologically different α-syn aggregation compared with those inoculated with the human WT fibril (hWT) strain. By using cryo-electron microscopy, we reveal at the near-atomic level that the hE46K strain induces both human and mouse WT α-syn monomers to form the fibril structure of the hE46K strain. Moreover, the induced hWT strain inherits most of the pathological traits of the hE46K strain as well. Our work suggests that the structural and pathological features of mutant strains could be propagated by the WT α-syn in such a way that the mutant pathology would be amplified in fPD.

α-Synuclein (α-Syn) is the main component of Lewy bodies, which serve as the common histological hallmark of Parkinson’s disease (PD) and other synucleinopathies (1, 2). α-Syn fibrillation and cell-to-cell transmission in the brain play essential roles in disease progression (3–5). Interestingly, WT α-syn could form fibrils with distinct polymorphs, which exhibit disparate seeding capability in vitro and induce distinct neuropathologies in mouse models (6–10). Therefore, it is proposed that α-syn fibril polymorphism may underlie clinicopathological variability of synucleinopathies (6, 9). In fPD, several single-point mutations of SNCA have been identified, which are linked to early-onset, severe, and highly heterogeneous clinical symptoms (11–13). These mutations have been reported to influence either the physiological or pathological function of α-syn (14). For instance, A30P weakens while E46K strengthens α-syn membrane binding affinity that may affect its function in synaptic vesicle trafficking (14, 15). E46K, A53T, G51D, and H50Q have been found to alter the aggregation kinetics of α-syn in different manners (15–17). Recently, several cryogenic electron microscopy (cryo-EM) studies revealed that α-syn with these mutations forms diverse fibril structures that are distinct from the WT α-syn fibrils (18–26). Whether and how hereditary mutations induced fibril polymorphism contributes to the early-onset and exacerbated pathology in fPD remains to be elucidated. More importantly, most fPD patients are heterozygous for SNCA mutations (12, 13, 27, 28), which leads to another critical question: could mutant fibrils cross-seed WT α-syn to orchestrate neuropathology in fPD patients?E46K mutation is one of the eight disease-causing mutations on SNCA originally identified from a Spanish family with autosomal-dominant PD (11). E46K-associated fPD features early-onset motor symptoms and rapid progression of dementia with Lewy bodies (11). Studies have shown that E46K mutant has higher neurotoxicity than WT α-syn in neurons and mouse models overexpressing α-syn (29–32). The underlying mechanism is debatable. Some reported that E46K promotes the formation of soluble species of α-syn without affecting the insoluble fraction (29, 30), while others suggested that E46K mutation may destabilize α-syn tetramer and induce aggregation (31, 32). Our previous study showed that E46K mutation disrupts the salt bridge between E46 and K80 in the WT fibril strain and rearranges α-syn into a different polymorph (33). Compared with the WT strain, the E46K fibril strain is prone to be fragmented due to its smaller and less stable fibril core (33). Intriguingly, the E46K strain exhibits higher seeding ability in vitro, suggesting that it might induce neuropathology different from the WT strain in vivo (33).In this study, we found that human E46K and WT fibril strains (referred to as hE46K and hWT strains) induced α-syn aggregates with distinct morphologies in mice. Mice injected with the hE46K strain developed more α-syn aggregation and early-onset motor deficits compared with the mice injected with the hWT strain. Notably, the hE46K strain was capable of cross-seeding both human and mouse WT (mWT) α-syn to form fibrils (named as hWT_cs and mWT_cs). The cross-seeded fibrils replicated the structure and seeding capability of the hE46K template both in vitro and in vivo. Our results suggest that the hE46K strain could propagate its structure as well as the seeding properties to the WT monomer so as to amplify the α-syn pathology in fPD.     

Molecular basis of the dual role of the Mlh1-Mlh3 endonuclease in MMR and in meiotic crossover formation

In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ’s dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.

During the first meiotic division, in most organisms, each pair of homologous chromosomes (homologs) needs to experience at least one crossover to ensure their accurate segregation and increase genetic diversity of the progeny (1, 2). Crossovers are generated after programmed DNA double-strand break (DSB) formation, and their subsequent repair by homologous recombination (3). Failure to achieve at least one crossover per homolog pair results in aneuploid gametes. These dysfunctions are frequent causes of spontaneous miscarriages and birth defects in humans (1). DSBs are generated by the meiosis-specific Spo11 protein and resected to form 3′ single-stranded tails that are directed to invade and pair with an unbroken homologous template, preferentially on the homolog (3). Invasion intermediates are a substrate for DNA synthesis. After the capture of the second DSB end, a subset of the intermediates is converted into double Holliday junctions (dHJs), which in meiosis are primarily resolved into crossovers (4, 5). The remaining recombination intermediates are repaired as noncrossovers.MutLγ (Mlh1-Mlh3 in yeast and MLH1-MLH3 in human) is essential for the formation of meiotic crossovers in many organisms. The MutLγ heterodimer possesses, similarly to MutLα (Mlh1-Pms1 in yeast, MLH1-PMS2 in human), a latent endonuclease activity (6–10). It has been proposed that the MutLγ endonuclease activity catalyzes the resolution of the dHJ intermediates and promotes the formation of crossovers (8, 11). In agreement with this, Mlh1 and Mlh3 form foci on pachytene chromosomes in different organisms at future crossover sites (12–14). In yeast mlh3 mutants, the crossover rates are reduced to 50 to 70% of the wild-type level (8, 15–17). These mutants exhibit failure in chromosome disjunction and consequently a decrease of spore viability. In Mlh3^-/- mice, males and females present a crossover defect that leads to aneuploidy (18). In agreement with the proposed resolvase role of MutLγ, a mutant of the active site within the conserved DQHAX₂EX₄E motif of Saccharomyces cerevisiae Mlh3, D523N, results in a loss of activity and confers a phenotype similar to the mlh3∆ mutant with decreased crossover frequencies (8). A mutation in the endonuclease domain of mouse MLH3 leads to infertile males and strongly reduced crossover numbers, despite a correct loading of factors essential for crossover resolution (19). In budding yeast and mammals, MLH1 deletion is also associated with severe dysfunctions in meiosis due to crossover defects, combined with high genetic instability due to its additional central role with Pms1 (as part of the MutLα complex) in mutation avoidance (20).In budding yeast, the majority of meiotic crossovers are formed by MutLγ, requiring in addition other proteins including the ZMM proteins (Zip1-4, Spo16, Mer3, and Msh4-Msh5), Exo1, and the Sgs1-Top3-Rmi1 complex (11, 21). The ZMM factors are proposed to stabilize the recombination intermediates and protect them from the action of helicases (reviewed in ref. 22). The 5′-3′ exonuclease, Exo1, is important for crossover formation independently of its nuclease activity and likely acts as scaffold factor in particular through its interaction motif with Mlh1 (17). We recently reported that MutLγ-Exo1 associates with recombination intermediates, followed by direct Cdc5 recruitment that triggers MutLγ crossover activity (23). Despite its central role in the formation of crossovers, to date, the MutLγ endonuclease does not show any specific activity on either a single HJ or dHJ DNA substrate in vitro, in contrast to other resolvases or structure-specific endonucleases (24). Recently, it was shown that human MutLγ is part of an ensemble with MutSγ, EXO1, RFC, and PCNA that preferentially cleaves plasmid DNA containing a HJ, although it does not cleave symmetrically across the junction, as would be expected for a canonical resolvase (25, 26).MutLγ is also an accessory factor of the postreplicative mismatch repair (MMR). In S. cerevisiae, MutLα is the major MutL homolog involved in MMR. It is targeted to DNA mismatches by the MutS homologs and introduces DNA nicks to initiate the excision of the strand containing the mismatch. MutLα contains an endonuclease site formed by three conserved motifs in Pms1 and the last conserved amino acids of Mlh1 (6, 7, 27). Mutation of this active site (e.g., pms1E707K in yeast) is associated with high mutations rates. A minor role of MutLγ has been reported in the repair of a subset of DNA mismatches recognized by Msh2-Msh3 (28, 29). The third MutL heterodimer, Mlh1-Mlh2 (MLH1-PMS1 in mammals) or MutLβ, has no endonuclease motif or activity. In yeast, mlh2Δ cells present only a slight defect in the repair of a subset of frameshift mutations (30–32). Apart from this function in MMR, we recently identified an interaction of MutLβ with Mer3 helicase that limits gene conversion tract lengths (31). A major challenge is to further characterize the molecular basis of the specific interactions and regulations of the three MutL homologs with their DNA substrates and protein partners in MMR and in meiotic recombination.MutLγ and MutLα present an overall common organization with a N-terminal domain (NTD) bearing an ATPase function, connected through a long linker to a C-terminal domain (CTD). The CTD mediates the dimerization of the complexes (33, 34) and possesses the endonuclease site (6–8). Upon ADP and ATP binding, the NTD undergoes large asymmetric conformational changes that can position it into a close proximity to the CTD (35, 36). The heterodimers MutLα and MutLγ can slide on double-stranded DNA (dsDNA) with linear diffusion modes (37, 38) and control strand excision during MMR (39). However, both heterodimers differ in their DNA-binding properties. MutLα has very low affinity for short dsDNA in physiological salt concentrations and can bind cooperatively to long dsDNA (>200 bp), forming long continuous protein tracts (40). In contrast, MutLγ binds short-branched DNA substrates with a higher affinity (in the nanomolar range) and with a marked preference for HJs (9). MutLγ also binds, albeit more weakly, short and long dsDNA and is proposed to form oligomers on long dsDNA (9, 41). The Pms1 and Mlh3 subunits present a moderate sequence identity precluding correct modeling of MutLγ from the MutLα crystal structures and thus limiting our understanding of the molecular basis of the specificity of MutLα and MutLγ heterodimers in MMR and meiosis, respectively.Here, we present the crystal structure of the S. cerevisiae MutLγ(CTD), and we compare it with the three-dimensional structure of the S. cerevisiae MutLα(CTD) that we previously reported (27). We reveal differences between the two complexes with regard to the size of the heterodimerization interface, the regulatory domain position, and the shape of the cavity surrounding the endonuclease site. We characterize the role of the last amino acids of Mlh1 using mutant alleles and identify a central role of the last three conserved residues of Mlh1 in vivo for both MMR and meiotic recombination. We analyze the DNA-binding specificities of the MutLα(CTD) and Mut-Lγ(CTD) and compare to the properties of full-length proteins. We then identify positions in Mlh3(NTD) and Mlh3(CTD) that can participate to the positioning of the NTD close to the CTD and characterize the corresponding mutants in meiosis. Finally, we report a filament arrangement of the MutLγ(CTD) in the crystal in agreement with oligomers proposed in previous studies (25, 41), and we characterize an allele mutated in this oligomerization region.     

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.     

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

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

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

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

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

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