Naphthylphthalamic acid associates with and inhibits PIN auxin transporters

N-1-naphthylphthalamic acid (NPA) is a key inhibitor of directional (polar) transport of the hormone auxin in plants. For decades, it has been a pivotal tool in elucidating the unique polar auxin transport-based processes underlying plant growth and development. Its exact mode of action has long been sought after and is still being debated, with prevailing mechanistic schemes describing only indirect connections between NPA and the main transporters responsible for directional transport, namely PIN auxin exporters. Here we present data supporting a model in which NPA associates with PINs in a more direct manner than hitherto postulated. We show that NPA inhibits PIN activity in a heterologous oocyte system and that expression of NPA-sensitive PINs in plant, yeast, and oocyte membranes leads to specific saturable NPA binding. We thus propose that PINs are a bona fide NPA target. This offers a straightforward molecular basis for NPA inhibition of PIN-dependent auxin transport and a logical parsimonious explanation for the known physiological effects of NPA on plant growth, as well as an alternative hypothesis to interpret past and future results. We also introduce PIN dimerization and describe an effect of NPA on this, suggesting that NPA binding could be exploited to gain insights into structural aspects of PINs related to their transport mechanism.

Many aspects of plant growth are controlled by the hormone auxin. A distinct feature of auxin is that its hormonal action requires it to be actively transported between cells and ultimately throughout the whole plant in a controlled directional or polarized manner, a process known as polar auxin transport (PAT). The ability of plants to perform PAT is ascribed to the auxin export activity of PIN transporters (1). Plasma membrane PINs can be restricted to a specific side of cells (2), and when this polarity is maintained in continuous plant cell files, the combined activity of identically localized PINs results in auxin flowing in that direction (3). This lays the vectorial foundations for PAT to create local auxin gradients and plant-wide PAT streams that are critical for auxin action and normal plant growth (4, 5).Synthetic PAT inhibitors such as N-1-naphthylphthalamic acid (NPA) were initially developed as herbicides and then subsequently exploited by researchers to identify and characterize the unique PAT-based mechanisms that drive plant development (6). Having been used for over six decades, the question as to how NPA actually inhibits PAT has been keenly pursued. Several putative modes of action have been proposed, but the topic remains to date not fully or satisfactorily resolved (6).Early studies established NPA binding with high affinity to membrane-integral components of plant membranes (7–10). With the later discovery of pin1 mutants bearing their distinct bare inflorescences reminiscent of NPA-treated plants (11), followed by identification of the PIN gene family and gradual confirmation that PINs were NPA-sensitive auxin transporters that mediated PAT (1–5), it was apparent that the physiological and genetic evidence overwhelmingly linked NPA to inhibition of PIN activity (6). However, direct molecular association of NPA with PINs has never been reported (6). Instead, a substantial body of data has accumulated suggesting that the NPA target is not PIN itself, but rather other proteins or complexes that either actively coparticipate in PAT or are indirectly involved in control of PAT components (6, 12). Members of the B-family of ABC transporters, such as ABCB1 and ABCB19, showed high-affinity NPA binding and NPA-sensitive auxin export (1, 12–15), thus leading to proposals that they may either physically interact with PINs, or functionally interact such that their nonpolar auxin export activity contributes to PAT and/or to regulation of PINs (12, 16). In these scenarios, PIN/PAT would be rendered vulnerable to the NPA sensitivity of ABCB. However, these schemes are not yet fully resolved, are not fully consistent with key genetic and physiological data (6), and are particularly obfuscated by ABCB1/19 functioning both interactively and independently from PINs (1, 12, 15–20), with ABCB-PIN interaction occurring in an as-yet-unclarified manner (15, 18).A further twist in assigning ABCBs as the main NPA target is their regulation by their chaperone TWD1/FKBP42 (14, 16), with TWD1 itself also being an NPA-binding protein (14, 17). NPA interferes with this regulation and affects TWD1-ABCB interaction, but curiously NPA cannot bind stably to the ABCB-TWD1 complex (14, 17). As TWD1 has also been implicated in NPA-sensitive actin-based PIN trafficking (17), this has led to a model proposing that TWD1 could mediate the NPA sensitivities of both ABCB and PINs, thus presenting TWD1 as a modulator of PAT (17, 21). In an analogous scheme in some plant species, CYPA immunophilins such as tomato DGT, which are functionally similar to TWD1/FKBP42, are suggested to replace TWD1 in modulating auxin transporters and transducing NPA effects to PINs (12, 21).Similar to TWD1, BIG/TIR3 has also been associated with NPA and PIN trafficking (22). Given the undisputed role of trafficking in controlling PIN polarity (5), these reported effects warrant attention, although they are inconsistent with other reports that NPA perturbs neither vesicular trafficking nor actin dynamics in conditions where auxin transport is inhibited (23, 24). Together with trafficking, phosphorylation is another key modulator of PIN polarity as well as activity (5), so it is not surprising to find hypotheses suggesting that NPA could interfere with critical phosphorylation events (6), particularly as PID, a kinase crucial for PIN trafficking and activation, has also been connected to ABCB function and TWD1/ABCB/NPA interactions (25). Others propose that NPA may mimic natural compounds in their capacity as endogenous regulators of PAT, with plant flavonoids being suspected candidates (6, 26). Since flavonoids can compete with or inhibit ATP-binding in mammalian kinases and ABC transporters (27, 28), and as flavonoids can bind to and inhibit PID (25), a phosphorylation-based NPA mode of action would overlap with this hypothesis and poses the question whether NPA acts similarly as an ATP mimic.With these many potential NPA-affected pathways, there is a need to distinguish between low- and high-affinity NPA targets and possible secondary effects due to prolonged PAT inhibition. Current consensus is that low concentrations of NPA (<10 µM) cause direct inhibition of auxin transporters in PAT (21) and the consequent physiological effects seen in planta (IC₅₀ 0.1 to 10 µM) (7, 9, 19, 23, 29). This is associated with high-affinity binding to membranes (K_d 0.01 to 0.1 µM) (7, 8) and the inhibition of PIN/ABCB activity in short-term auxin transport assays (1, 14, 18, 20, 23). In contrast, NPA is thought to affect trafficking (21, 30) and other non-PAT processes (31) when used at higher doses (50 to 200 µM NPA), presumably via binding to its lower-affinity targets, although excessive NPA exposure may also have fast-acting toxic side effects (23). As the in vitro affinity of TWD1 for NPA is surprisingly low (K_d ∼100 µM) (17), the TWD1-mediated NPA effects on PIN/PAT are thought to be of the low-affinity type and linked to trafficking perturbations (17, 21). However, as NPA is always externally applied to plants or cells, it is not clear how or where the drug distributes or accumulates, and thus there may be discrepancies between actual and reported/apparent effective concentrations, as might be the case for TWD1 (17). Finally, NPA also binds with low affinity to inhibit APM1, an aminopeptidase implicated in auxin-related plant growth, but as with trafficking effects, this low-affinity NPA interaction is not connected to direct regulation of PAT (31).Thus, the available data proffer various indirect mechanisms that could lead to NPA inhibition of PIN-mediated PAT, but the proposed schemes have complicating aspects and struggle at times to satisfactorily explain the prime effects of NPA. Here we propose an alternative simpler scenario involving a more direct link between NPA and PINs that would resolve some of these currently outstanding issues. We present evidence from heterologous transport assays, classical in situ membrane binding, and oligomerization studies which collectively suggest that NPA can interact directly in a high-affinity manner with PINs, leading to conformational or structural effects and inhibition of auxin export activity.     

The Arabidopsis NRT1/PTR FAMILY protein NPF7.3/NRT1.5 is an indole-3-butyric acid transporter involved in root gravitropism

Active membrane transport of plant hormones and their related compounds is an essential process that determines the distribution of the compounds within plant tissues and, hence, regulates various physiological events. Here, we report that the Arabidopsis NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY 7.3 (NPF7.3) protein functions as a transporter of indole-3-butyric acid (IBA), a precursor of the major endogenous auxin indole-3-acetic acid (IAA). When expressed in yeast, NPF7.3 mediated cellular IBA uptake. Loss-of-function npf7.3 mutants showed defective root gravitropism with reduced IBA levels and auxin responses. Nevertheless, the phenotype was restored by exogenous application of IAA but not by IBA treatment. NPF7.3 was expressed in pericycle cells and the root tip region including root cap cells of primary roots where the IBA-to-IAA conversion occurs. Our findings indicate that NPF7.3-mediated IBA uptake into specific cells is required for the generation of appropriate auxin gradients within root tissues.

Plant hormones are vital compounds that function as signaling molecules and control a wide range of physiological responses so that plants can cope with fluctuating environments (1). Often, plant hormones induce physiological responses locally (2–6); thus, the endogenous levels as well as the signal transduction pathways of plant hormones are regulated very precisely. Endogenous concentrations of bioactive plant hormones are primarily determined by the balance between the rates of biosynthesis and degradation (or inactivation). Additionally, specific transport systems for plant hormones and their related compounds are also considered to be critical components for generating appropriate hormone concentrations within cells, since hormone biosynthesis and perception do not necessarily take place in the same cell. In fact, movement of plant hormones and their precursors has been reported (7).Auxin is the best-characterized mobile plant hormone that plays crucial roles in many aspects of plant growth and development by mainly regulating cell division, cell elongation, and cell differentiation (8). Indole-3-acetic acid (IAA) is the major naturally occurring auxin. Measurements of IAA by mass spectrometry as well as visualization of auxin responses by reporter genes have suggested that IAA is differentially distributed within plant tissues (9). Furthermore, appropriate IAA gradients or local IAA maxima/minima are required for proper auxin functions (9). In fact, mutants impaired in IAA transport have been isolated based on their defects in organ development and various physiological responses (9). Evidence for IAA transport in a cell-to-cell manner by combinations of several transmembrane transporters is well documented. The plant-specific PIN-FORMED (PIN) transporters and the AUX1/LAX family of amino acid permease-like proteins are IAA efflux and influx carriers, respectively (10, 11). PINs determine the direction of IAA flow by localizing unevenly in the plasma membrane, whereas polarity is not observed for AUX/LAXs distribution (9). Also, some subfamily B members of the adenosine 5′-triphosphate-binding cassette (ABC) transporter (ABCBs) family act as IAA transporters (9).IAA is mainly synthesized from tryptophan via indole-3-pyruvic acid (12); however, minor routes for IAA biosynthesis have also been suggested (13). Indole-3-butyric acid (IBA) has auxin activity when applied exogenously to plants. Arabidopsis mutants isolated based on their resistance to exogenous IBA are defective in the conversion of IBA to IAA (14). Since IBA has been detected in several plant species (13), the compound is considered to be an endogenous IAA precursor. Possible IBA transporters have been identified (15–17), suggesting that IBA transport is also an important factor that regulates endogenous IAA levels.NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY (NPF) proteins have been characterized as transporters for several plant hormones including auxin, although the family was initially identified as nitrate or di/tripeptide transporters (18, 19). The Arabidopsis NPF6.3 protein (also called as CHL1 or NRT1.1) is a well-characterized dual-affinity nitrate transporter that mediates nitrogen uptake from the rhizosphere (20, 21); however, the same protein also transports IAA (22). More recently, the Arabidopsis NPF5.12 protein, also named TRANSPORTER OF IBA1 (TOB1), was identified as an IBA transporter that localizes to the vacuolar membrane (17). Here, we report that NPF7.3, originally identified as a low-affinity nitrate transporter (NRT1.5) and later shown to be a proton/potassium antiporter in Arabidopsis (23, 24), functions as an IBA transporter. We found that npf7.3 mutant roots were not able to respond properly to gravity. Our transport assays in yeast demonstrated that NPF7.3 efficiently mediated IBA uptake into the cells. Asymmetric distribution of auxin activities in root tips following the reorientation of roots was not observed in the npf7.3 mutants, and the defect was restored by IAA but not by IBA treatment. These results suggest that cellular IBA uptake mediated by NPF7.3 contributes to the production of IAA required to fully induce Arabidopsis root gravitropism.     

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.     

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.     

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

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

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

SLFN11 promotes CDT1 degradation by CUL4 in response to replicative DNA damage,while its absence leads to synthetic lethality with ATR/CHK1 inhibitors

Schlafen-11 (SLFN11) inactivation in ∼50% of cancer cells confers broad chemoresistance. To identify therapeutic targets and underlying molecular mechanisms for overcoming chemoresistance, we performed an unbiased genome-wide RNAi screen in SLFN11-WT and -knockout (KO) cells. We found that inactivation of Ataxia Telangiectasia- and Rad3-related (ATR), CHK1, BRCA2, and RPA1 overcome chemoresistance to camptothecin (CPT) in SLFN11-KO cells. Accordingly, we validate that clinical inhibitors of ATR (M4344 and M6620) and CHK1 (SRA737) resensitize SLFN11-KO cells to topotecan, indotecan, etoposide, cisplatin, and talazoparib. We uncover that ATR inhibition significantly increases mitotic defects along with increased CDT1 phosphorylation, which destabilizes kinetochore-microtubule attachments in SLFN11-KO cells. We also reveal a chemoresistance mechanism by which CDT1 degradation is retarded, eventually inducing replication reactivation under DNA damage in SLFN11-KO cells. In contrast, in SLFN11-expressing cells, SLFN11 promotes the degradation of CDT1 in response to CPT by binding to DDB1 of CUL4^CDT2 E3 ubiquitin ligase associated with replication forks. We show that the C terminus and ATPase domain of SLFN11 are required for DDB1 binding and CDT1 degradation. Furthermore, we identify a therapy-relevant ATPase mutant (E669K) of the SLFN11 gene in human TCGA and show that the mutant contributes to chemoresistance and retarded CDT1 degradation. Taken together, our study reveals new chemotherapeutic insights on how targeting the ATR pathway overcomes chemoresistance of SLFN11-deficient cancers. It also demonstrates that SLFN11 irreversibly arrests replication by degrading CDT1 through the DDB1–CUL4^CDT2 ubiquitin ligase.

Schlafen-11 (SLFN11) is an emergent restriction factor against genomic instability acting by eliminating cells with replicative damage (1–6) and potentially acting as a tumor suppressor (6, 7). SLFN11-expressing cancer cells are consistently hypersensitive to a broad range of chemotherapeutic drugs targeting DNA replication, including topoisomerase inhibitors, alkylating agents, DNA synthesis, and poly(ADP-ribose) polymerase (PARP) inhibitors compared to SLFN11-deficient cancer cells, which are chemoresistant (1, 2, 4, 8–17). Profiling SLFN11 expression is being explored for patients to predict survival and guide therapeutic choice (8, 13, 18–24).The Cancer Genome Atlas (TCGA) and cancer cell databases demonstrate that SLFN11 mRNA expression is suppressed in a broad fraction of common cancer tissues and in ∼50% of all established cancer cell lines across multiple histologies (1, 2, 5, 8, 13, 25, 26). Silencing of the SLFN11 gene, like known tumor suppressor genes, is under epigenetic mechanisms through hypermethylation of its promoter region and activation of histone deacetylases (HDACs) (21, 23, 25, 26). A recent study in small-cell lung cancer patient-derived xenograft models also showed that SLFN11 gene silencing is caused by local chromatin condensation related to deposition of H3K27me3 in the gene body of SLFN11 by EZH2, a histone methyltransferase (11). Targeting epigenetic regulators is therefore an attractive combination strategy to overcome chemoresistance of SLFN11-deficient cancers (10, 25, 26). An alternative approach is to attack SLFN11-negative cancer cells by targeting the essential pathways that cells use to overcome replicative damage and replication stress. Along these lines, a prior study showed that inhibition of ATR (Ataxia Telangiectasia- and Rad3-related) kinase reverses the resistance of SLFN11-deficient cancer cells to PARP inhibitors (4). However, targeting the ATR pathway in SLFN11-deficient cells has not yet been fully explored.SLFN11 consists of two functional domains: A conserved nuclease motif in its N terminus and an ATPase motif (putative helicase) in its C terminus (2, 6). The N terminus nuclease has been implicated in the selective degradation of type II tRNAs (including those coding for ATR) and its nuclease structure can be derived from crystallographic analysis of SLFN13 whose N terminus domain is conserved with SLFN11 (27, 28). The C terminus is only present in the group III Schlafen family (24, 29). Its potential ATPase activity and relationship to chemosensitivity to DNA-damaging agents (3–5) imply that the ATPase/helicase of SLFN11 is involved specifically in DNA damage response (DDR) to replication stress. Indeed, inactivation of the Walker B motif of SLFN11 by the mutation E669Q suppresses SLFN11-mediated replication block (5, 30). In addition, SLFN11 contains a binding site for the single-stranded DNA binding protein RPA1 (replication protein A1) at its C terminus (3, 31) and is recruited to replication damage sites by RPA (3, 5). The putative ATPase activity of SLFN11 is not required for this recruitment (5) but is required for blocking the replication helicase complex (CMG-CDC45) and inducing chromatin accessibility at replication origins and promoter sites (5, 30). Based on these studies, our current model is that SLFN11 is recruited to “stressed” replication forks by RPA filaments formed on single-stranded DNA (ssDNA), and that the ATPase/helicase activity of SLFN11 is required for blocking replication progression and remodeling chromatin (5, 30). However, underlying mechanisms of how SLFN11 irreversibly blocks replication in DNA damage are still unclear.Increased RPA-coated ssDNA caused by DNA damage and replication fork stalling also triggers ATR kinase activation, promoting subsequent phosphorylation of CHK1, which transiently halts cell cycle progression and enables DNA repair (32). ATR inhibitors are currently in clinical development in combination with DNA replication damaging drugs (33, 34), such as topoisomerase I (TOP1) inhibitors, which are highly synergistic with ATR inhibitors in preclinical models (35). ATR inhibitors not only inhibit DNA repair, but also lead to unscheduled replication origin firing (36), which kills cancer cells (37, 38) by inducing genomic alterations due to faulty replication and mitotic catastrophe (33).The replication licensing factor CDT1 orchestrates the initiation of replication by assembling prereplication complexes (pre-RC) in G1-phase before cells enter S-phase (39). Once replication is started by loading and activation of the MCM helicase, CDT1 is degraded by the ubiquitin proteasomal pathway to prevent additional replication initiation and ensure precise genome duplication and the firing of each origin only once per cell cycle (39, 40). At the end of G2 and during mitosis, CDT1 levels rise again to control kinetochore-microtubule attachment for accurate chromosome segregation (41). Deregulated overexpression of CDT1 results in rereplication, genome instability, and tumorigenesis (42). The cellular CDT1 levels are tightly regulated by the damage-specific DNA binding protein 1 (DDB1)–CUL4^CDT2 E3 ubiquitin ligase complex in G1-phase (43) and in response to DNA damage (44, 45). How CDT1 is recognized by CUL4^CDT2 in response to DNA damage remains incompletely known.In the present study, starting with a human genome-wide RNAi screen, bioinformatics analyses, and mechanistic validations, we explored synthetic lethal interactions that overcome the chemoresistance of SLFN11-deficient cells to the TOP1 inhibitor camptothecin (CPT). The strongest synergistic interaction was between depletion of the ATR/CHK1-mediated DNA damage response pathways and DNA-damaging agents in SLFN11-deficient cells. We validated and expanded our molecular understanding of combinatorial strategies in SLFN11-deficient cells with the ATR (M4344 and M6620) and CHK1 (SRA737) inhibitors in clinical development (33, 46, 47) and found that ATR inhibition leads to CDT1 stabilization and hyperphosphorylation with mitotic catastrophe. Our study also establishes that SLFN11 promotes the degradation of CDT1 by binding to DDB1, an adaptor molecule of the CUL4^CDT2 E3 ubiquitin ligase complex, leading to an irreversible replication block in response to replicative DNA damage.     

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.     

Conditional destabilization of the TPLATE complex impairs endocytic internalization

In plants, endocytosis is essential for many developmental and physiological processes, including regulation of growth and development, hormone perception, nutrient uptake, and defense against pathogens. Our toolbox to modulate this process is, however, rather limited. Here, we report a conditional tool to impair endocytosis. We generated a partially functional TPLATE allele by substituting the most conserved domain of the TPLATE subunit of the endocytic TPLATE complex (TPC). This substitution destabilizes TPC and dampens the efficiency of endocytosis. Short-term heat treatment increases TPC destabilization and reversibly delocalizes TPLATE from the plasma membrane to aggregates in the cytoplasm. This blocks FM uptake and causes accumulation of various known endocytic cargoes at the plasma membrane. Short-term heat treatment therefore transforms the partially functional TPLATE allele into an effective conditional tool to impair endocytosis. Next to their role in endocytosis, several TPC subunits are also implicated in actin-regulated autophagosomal degradation. Inactivating TPC via the WDX mutation, however, does not impair autophagy, thus enabling specific and reversible modulation of endocytosis in planta.

Endocytosis is an evolutionarily conserved eukaryotic pathway by which extracellular material and plasma membrane (PM) components are internalized via vesicles (1, 2). Clathrin-mediated endocytosis (CME), relying on the scaffolding protein clathrin, is the most prominent and the most studied endocytic pathway (3–5). As clathrin does not interact directly with the PM, nor does it recognize cargoes, adaptor proteins are required to act as essential links between the clathrin coat and the PM (6). In plant cells, material selected for CME is recognized by two adaptor complexes, the adaptor complex 2 (AP-2) and the TPLATE complex (TPC) (7–9). In contrast to TPC, single subunit mutants of AP-2 are viable (7, 8, 10–13) and AP-2 recruitment and dynamics appear to rely on TPC function (8, 14).TPC represents an ancestral adaptor complex, which is however absent in present-day metazoans and yeasts. It was experimentally identified as an octameric complex in Arabidopsis and as a hexametric complex in Dictyostelium (8, 15). Plants, however, are the only eukaryotic supergroup identified so far where TPC is essential for life (8, 15), as knockout or severe knockdown of single subunits of TPC in Arabidopsis leads to pollen or seedling lethality, respectively (8, 13). Two TPC subunits, AtEH1/Pan1 and AtEH2/Pan1, were not associated with the other TPC core components when the complex was forced into the cytoplasm by truncating the TML subunit and did not copurify with the other TSET components in Dictyostelium. This indicates that they may be auxiliary components to the core TPC (8, 15). These AtEH/Pan1 proteins were recently identified as important players in actin-regulated autophagy in plants. AtEH/Pan1 proteins recruit several components of the endocytic machinery to the autophagosomes, and are degraded together with them under stress conditions (16). However, whether this pathway serves to degrade specific cargoes or to regulate the endocytic machinery itself (17), and whether the whole TPC is required for this degradation pathway, remains unclear.Genetic and chemical tools to manipulate endocytosis have been extensively investigated via interfering with the functions of endocytic players, such as clathrin (18–22), adaptor proteins (7, 10–12, 14, 23–25), and dynamin-related proteins (26–30). The chemical inhibitors originally used to affect CME in plants have recently been described to possess undesirable side effects (31) or to affect proteins that are not only specific for endocytosis: for example, clathrin itself, as it is also involved in TGN trafficking (19, 22). The same is true for several genetic tools currently available to affect CME in plants (18, 21, 22, 30). Manipulation of TPC, functioning exclusively at the PM, represents a very good candidate to affect CME more specifically. So far however, there are no chemical tools to target TPC functions or dominant-negative mutants available. Inducible silencing works, but causes seedling lethality and takes several days to become effective (8). The only tools to manipulate TPC function in viable plants consist of knock-down mutants with very mild reduction of expression and consequently similar mild effects on CME (8, 14, 16, 32).     

Macrophage LC3-associated phagocytosis is an immune defense against Streptococcus pneumoniae that diminishes with host aging

Streptococcus pneumoniae is a leading cause of pneumonia and invasive disease, particularly, in the elderly. S. pneumoniae lung infection of aged mice is associated with high bacterial burdens and detrimental inflammatory responses. Macrophages can clear microorganisms and modulate inflammation through two distinct lysosomal trafficking pathways that involve 1A/1B-light chain 3 (LC3)-marked organelles, canonical autophagy, and LC3-associated phagocytosis (LAP). The S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers an autophagic response in nonphagocytic cells, but the role of LAP in macrophage defense against S. pneumoniae or in age-related susceptibility to infection is unexplored. We found that infection of murine bone-marrow-derived macrophages (BMDMs) by PLY-producing S. pneumoniae triggered Atg5- and Atg7-dependent recruitment of LC3 to S. pneumoniae-containing vesicles. The association of LC3 with S. pneumoniae-containing phagosomes required components specific for LAP, such as Rubicon and the NADPH oxidase, but not factors, such as Ulk1, FIP200, or Atg14, required specifically for canonical autophagy. In addition, S. pneumoniae was sequestered within single-membrane compartments indicative of LAP. Importantly, compared to BMDMs from young (2-mo-old) mice, BMDMs from aged (20- to 22-mo-old) mice infected with S. pneumoniae were not only deficient in LAP and bacterial killing, but also produced higher levels of proinflammatory cytokines. Inhibition of LAP enhanced S. pneumoniae survival and cytokine responses in BMDMs from young but not aged mice. Thus, LAP is an important innate immune defense employed by BMDMs to control S. pneumoniae infection and concomitant inflammation, one that diminishes with age and may contribute to age-related susceptibility to this important pathogen.

Streptococcus pneumoniae (pneumococcus) commonly colonizes the nasopharynx asymptomatically but is also capable of infecting the lower respiratory tract to cause pneumonia and spreading to the bloodstream to cause septicemia and meningitis (1). Susceptibility to pneumonia and invasive disease caused by S. pneumoniae is remarkably higher in individuals aged 65 and over, leading to high rates of mortality and morbidity in the elderly population (1, 2). In countries, such as the United States and Japan, deaths due to pneumococcal pneumonia have been on the rise in parallel with the rapid growth in the elderly population (3, 4).A hallmark of pneumococcal pneumonia is a rapid and exuberant response by immune cells, such as neutrophils and macrophages. This innate immune response to S. pneumoniae lung infection is critical for pathogen clearance and the control of disease (5–7). Deficiencies in the number or function of innate phagocytic cells, such as neutropenia (8) or macrophage phagocytic receptor defects (9–12), lead to diminished pneumococcal clearance and increased risk of invasive pneumococcal disease in both mouse models and humans. Phagocytic activity in alveolar macrophages is important during early responses to subclinical infections (13–15), and during moderate S. pneumoniae lung infection, newly generated monocytes eggress from the bone marrow and migrate into the lungs, differentiating into monocyte-derived alveolar macrophages (16). In addition to directly eliminating the invading microbe, macrophages secrete key cytokines, such as tumor necrosis factor (TNF), interleukin-1β (IL-1β), and interleukin-6 (IL-6), that regulate effector cell functions and pulmonary inflammation (17–19).Although an innate immune response is critical for pathogen clearance, poorly controlled inflammation can lead to tissue damage and mortality (20, 21). For example, in murine models, neutrophilic infiltration can enhance pulmonary damage and disrupt epithelial barrier function, leading to bacteremia and mortality (22–25). Macrophages are critical not only in regulating the early inflammatory response, but are also crucial for curtailing inflammation during the resolution phase of infection to limit tissue damage and promote healing (26, 27).Elderly individuals have higher baseline and induced levels of inflammation, a phenomenon termed inflammaging (28), that contributes to many age-associated pathological conditions, including increased susceptibility to a variety of infectious diseases, such as S. pneumoniae infection (7, 28–30). S. pneumoniae-induced inflammation, characterized by increased levels of chemokines, proinflammatory cytokines, and decreased anti-inflammatory cytokines, such as IL-10, is enhanced in the elderly (29, 31) as well as in aged mice (32, 33) and correlates with ineffective immune responses. Age-related chronic exposure to TNF-α, for instance, dampens macrophage-mediated S. pneumoniae clearance during lung infection (34), and NLR family pyrin domain containing 3 inflammasome activation in macrophages diminishes upon aging in mice (35). However, the age-related changes in macrophage effector functions leading to diminished clearance of S. pneumoniae are incompletely understood.One important means of macrophage-mediated pathogen clearance is1A/1B-light chain-3 (LC3)-associated phagocytosis (LAP), a process by which cells target phagocytosed extracellular particles for efficient degradation (36–38). LAP combines the molecular machineries of phagocytosis and autophagy, resulting in the conjugation of the autophagic marker, the microtubule-associated protein LC3, to phosphatidylethanolamine on the phagosomal membrane, generating so-called “LAPosomes” that undergo facilitated fusion with lysosomes (38, 39). Canonical autophagy targets cytoplasmic components, such as damaged subcellular organelles and intracellular microbes for sequestration into double-membrane autophagic vesicles (40, 41). In contrast, LAPosomes retain the single-membrane nature of phagosomes, and their formation requires overlapping but nonidentical genes compared to canonical autophagy (42). In addition to enabling efficient degradation of phagocytosed bacteria, LAP also plays important immune regulatory roles, such as in curtailing proinflammatory cytokine production during the subsequent innate immune response (39, 43). Indeed, the LAP-mediated microbial defense and immunomodulatory functions work together to limit tissue damage and restore homeostasis (38).S. pneumoniae triggers canonical autophagy in epithelial cells and fibroblasts, and bacteria can be found in double-membrane vacuoles whose formation is dependent on autophagic machinery (44). Many bacterial pathogens that induce autophagy produce pore-forming toxins, which can damage endosomal membranes, thus, recruiting autophagic machinery to engulf injured organelles (45). Pneumococcus-induced autophagy is dependent on the cholesterol-dependent pore-forming toxin pneumolysin (PLY), which triggers the autophagic delivery of S. pneumoniae to lysosomes and results in bacterial killing (44, 46). Recently, a kinetic examination of S. pneumoniae-targeting autophagy in fibroblasts demonstrated that canonical autophagy was preceded by early and rapid PLY-dependent LAP (47). However, the requirements for this process were somewhat different from LAP in macrophages, and the pneumococcus-containing LAPosomes did not promote bacterial clearance but required subsequent transition to canonical autophagy to reduce bacterial numbers (46, 47).In the current study, we found that S. pneumoniae infection of murine bone-marrow-derived macrophages (BMDMs) induces LAP in a PLY-dependent manners and that age-related defects in BMDM LAP contributed to diminished bactericidal activity and enhanced proinflammatory cytokine production. Our results suggest that PLY-induced LAP promotes bacterial clearance, and age-associated dysregulation of this process may contribute to enhanced bacterial survival, poorly regulated inflammation, and increased susceptibility to invasive pneumococcal disease.     

Structure and activity of SLAC1 channels for stomatal signaling in leaves

Stomata in leaves regulate gas exchange between the plant and its atmosphere. Various environmental stimuli elicit abscisic acid (ABA); ABA leads to phosphoactivation of slow anion channel 1 (SLAC1); SLAC1 activity reduces turgor pressure in aperture-defining guard cells; and stomatal closure ensues. We used electrophysiology for functional characterizations of Arabidopsis thaliana SLAC1 (AtSLAC1) and cryoelectron microscopy (cryo-EM) for structural analysis of Brachypodium distachyon SLAC1 (BdSLAC1), at 2.97-Å resolution. We identified 14 phosphorylation sites in AtSLAC1 and showed nearly 330-fold channel-activity enhancement with 4 to 6 of these phosphorylated. Seven SLAC1-conserved arginines are poised in BdSLAC1 for regulatory interaction with the N-terminal extension. This BdSLAC1 structure has its pores closed, in a basal state, spring loaded by phenylalanyl residues in high-energy conformations. SLAC1 phosphorylation fine-tunes an equilibrium between basal and activated SLAC1 trimers, thereby controlling the degree of stomatal opening.

The stomatal pore, formed by a pair of specialized guard cells in plant leaves, serves as the gateway for water transpiration and atmospheric CO₂ influx for photosynthesis (1, 2). These pores need to be tightly controlled, as inadequate CO₂ intake is harmful and excessive water loss is devastating for plants (3). The guard cells respond to a wide range of environmental stimuli, including high CO₂ levels, O₃, low air humidity, and drought, and transduce these signals into appropriate turgor pressure changes that can adjust stomatal pore aperture (4, 5). It is well established that turgor pressure of guard cells is regulated by ion transport across the membrane, notably anions and potassium ions (6–8).The phytohormone abscisic acid (ABA) plays a central role in controlling stomatal closure via activation of a complex signaling pathway that is mediated by receptors, kinase/phosphatases, and ion channels (5, 9). It has been known that the activation of a slow anion current in guard cells is a key event leading to stomatal closure (10–13). The corresponding gene, slow anion channel 1 (SLAC1), was discovered in genetic screens for O₃ or CO₂ sensitivity in Arabidopsis; when this particular channel was mutated out, the mutants showed impaired response to stimuli (14–16).Arabidopsis thaliana SLAC1 (AtSLAC1) contains 556 amino acid (aa) residues, with a conserved transmembrane domain (TMD) between hydrophilic tails at both the N and C termini (15). Within the ABA signaling pathway, SLAC1 channel activity is controlled by a protein kinase-phosphatase pair (OST1/ABI1) (17–19). In the absence of ABA, OST1 kinase is bound and inhibited by ABI1 phosphatase (20–22). Under drought condition, ABA is accumulated into guard cells and perceived by the PYR1 receptor (23, 24). This resulting hormone–receptor complex then binds to phosphatase ABI1 to form a ternary hormone–receptor–phosphatase complex (25–28), which activates OST1 kinase, and leads to phosphorylation of SLAC1 (17, 18). Subsequently, SLAC1 releases anions (Cl⁻ and NO₃⁻) from guard cells, leading to membrane depolarization. Depolarization in turn activates potassium channels to release K⁺ via K⁺ channels and drives water out of the cell, decreasing guard cell turgor to close down the stomatal pore (8).It has been shown that SLAC1 activation is due to phosphorylation at its N terminus (17, 18, 29–31). The phosphorylation of S120 by OST1 is essential but not sufficient for SLAC1 activation monitored in Xenopus oocytes (18). S59 has been shown to be the target for both OST1 and other kinases (29, 31). In another phosphoproteomic analysis, using the fragment SLAC1¹⁻¹⁸⁶ as substrate, S86 and S113 were also shown to be phosphorylated by OST1 (32). OST1 also phosphorylates SLAC1 at the C terminus (17), with T513 identified as a potential phosphorylation site for SLAC1 activation (31). Although multiple phosphorylation sites have been proposed, the underlying mechanism for SLAC1 activation remains elusive.Our previous study on a SLAC1 bacterial homolog from Haemophilus influenzae (HiTehA) revealed a superfamily of trimeric anion channels (33) and found each protomer gated by a highly conserved phenylalanine. We observed an unusual high-energy rotameric conformation for this gating phenylalanine (F262 in HiTehA), and further mutational study on the corresponding site in AtSLAC1 (F450) suggested a unique mechanism for channel activation (33). Nevertheless, since HiTehA shares a mere 19% in sequence identity with AtSLAC1 and lacks the substantial N and C termini that are essential for channel activation, questions remain about SLAC1 structure and its regulation by kinase phosphorylation (7).Here we describe the cryoelectron microscopy (cryo-EM) structure of SLAC1 from Brachypodium distachyon (BdSLAC1) at 2.97-Å resolution, and we further characterize the channel activity on Arabidopsis SLAC1. BdSLAC1 shares 63% overall sequence identity with AtSLAC1, with 73% for the pore-forming TMD and 48% for the regulatory N and C termini. As for the bacterial homolog, each BdSLAC1 TMD comprises five tandemly repeated helical hairpins that surround a transmembrane pore in each protomer of the trimeric assembly. The SLAC1 pores are electropositive, consistent with its function as an anion channel. The regulatory N-terminal domain (190 aa) and C-terminal domain (65 aa) are flexibly associated and not discernible in the structure.We employed a SLAC1::OST1 fusion design for examining SLAC1 phosphorylation, whereby we systematically characterized critical sites responsible for channel activation. Inspired by the structure, we also conducted mutagenesis on the highly conserved phenylalanine residues related to channel gating. Altogether, our structural and functional analyses provide insights into SLAC1 gating and activity modulation. These findings allow us to propose a mechanism for finely tuned SLAC1 control of the stomatal pore in response to environmental stimuli.