Persulfidation of ATG18a regulates autophagy under ER stress in Arabidopsis

Hydrogen sulfide (H₂S) is an endogenously generated gaseous signaling molecule, which recently has been implicated in autophagy regulation in both plants and mammals through persulfidation of specific targets. Persulfidation has been suggested as the molecular mechanism through which sulfide regulates autophagy in plant cells. ATG18a is a core autophagy component that is required for bulk autophagy and also for reticulophagy during endoplasmic reticulum (ER) stress. In this research, we revealed the role of sulfide in plant ER stress responses as a negative regulator of autophagy. We demonstrate that sulfide regulates ATG18a phospholipid-binding activity by reversible persulfidation at Cys103, and that this modification activates ATG18a binding capacity to specific phospholipids in a reversible manner. Our findings strongly suggest that persulfidation of ATG18a at C103 regulates autophagy under ER stress, and that the impairment of persulfidation affects both the number and size of autophagosomes.

Macroautophagy (hereafter referred to as autophagy, from the Greek meaning “self-eating”) is a major catabolic process in eukaryotic cells to degrade dysfunctional or unnecessary cellular components, either non-selectively or selectively (1, 2). It has conserved functions in development, cellular homeostasis, and stress responses from yeast to plants and mammals. In plants, autophagy is critically important in many aspects of plant life, including seedling establishment, development, stress resistance, metabolism, and reproduction (1). The autophagy mechanism involves the enclosure of a portion of the cytoplasm into a double membrane vesicle, named an autophagosome. The outer membrane of the autophagosome finally fuses with the vacuole (in yeast and plants) to release the inner autophagic body for hydrolytic degradation of the sequestered cargo. About 40 ATG (autophagy related) genes have been identified in Arabidopsis, which are required for autophagosome formation (3). Autophagy was initially characterized as a bulk degradation pathway induced by nutrient deprivation with a role in nutrient recycling to enable cell survival, but it also contributes to intracellular homeostasis by selectively degrading aggregated proteins, damaged mitochondria, ribosomes, toxic macromolecules, excess peroxisomes, and pathogens to prevent toxicity (4–6). In particular, although the endoplasmic reticulum (ER) is involved in autophagic processes as a source for membranes, it is also the target of a selective type of autophagy, termed reticulophagy or ER-phagy. In plants, this ER-phagy is induced in response to ER stress produced by tunicamycin (TM) or dithiothreitol (DTT) treatments (7) and upon starvation (8). Selective autophagy is mediated by the binding of adaptor proteins, which link a cargo targeted for degradation to the autophagosome machinery (9). These selective autophagy receptors share the feature of interacting with the autophagosome-localized protein ATG8 through an ATG8-interacting motif or a ubiquitin-interacting motif, leading to their recruitment into forming autophagosomes (10–12).An increasing number of targets for selective autophagy under different stress conditions have emerged in recent years, but the underlying mechanisms of regulation of their degradation are still so far unknown. The activation of bulk and selective autophagy must be tightly controlled by the cellular conditions. In that sense, ATG4 is the only ATG that has been shown to be redox regulated in animal, yeast, algae, and plant systems (13–18). Nevertheless, in the last decade, a growing number of targets involved in autophagy have been shown to be regulated by different posttranslational modifications (PTMs); for example, ATG4b and ATG1 are regulated by S‐nitrosylation and phosphorylation (19, 20). Therefore, the ability of the ATG proteins to interact with a number of autophagic regulators is modulated by different PTMs such as phosphorylation, glycosylation, ubiquitination, and S-nitrosylation (21).Protein persulfidation is another player in the redox regulation of certain proteins. It is the mechanism for sulfide-mediated signaling and is an oxidative posttranslational modification of cysteine residues caused by hydrogen sulfide (H₂S) in which thiolate (–SH) is transformed to a persulfide group (–SSH). Persulfidation of proteins can affect their function, localization inside the cells, stability, and resistance to oxidative stress (22–27). H₂S is an endogenously generated gaseous signaling molecule, which has been recently implicated in autophagy regulation both in plants and mammals (28–30).Analysis of the Arabidopsis des1 mutant, impaired in the cytosolic production of H₂S from cysteine, led to the conclusion that H₂S acts as an inhibitor of autophagy induced by nutrient deprivation (28). Interestingly, its action is independent of reactive oxygen species (ROS) and nitrogen starvation, and the mechanism of autophagy inhibition by H₂S has been proposed to be through persulfidation of specific targets (31). Recently, regulation of the proteolytic activity of ATG4 by persulfidation has been demonstrated in plants (16). Autophagy induced upon nitrogen starvation or osmotic stress was negatively regulated by sulfide, and the mechanism has been explained through persulfidation of C170 of this ATG4 protease, which inhibits proteolytic activity. Collectively, these results suggest that persulfidation may be the molecular mechanism through which sulfide regulates autophagy in plant cells. The susceptibility to persulfidation of the additional ATG-related proteins ATG18a, ATG3, ATG5, and ATG7 was revealed using a high throughput proteomic approach (32), although the role of this modification in these other ATG proteins has not yet been revealed. ATG18a is a core autophagy protein that binds to phosphoinositides (33, 34). It has a seven-bladed β-propeller structure formed by WD40 repeats that bind phosphatidylinositol 3-phosphate (PtdIns(3)P) or phosphatidylinositol (3, 5)-bisphosphate (PtdIns(3,5)P2). These two phosphoinositide-binding sites are located in blades five and six surrounding and sandwiching the conserved L/FRRG motif. ATG18a forms a complex with ATG2, which is involved in autophagosome biogenesis during phagophore expansion (34) and involved in the formation of preautophagosomal structures and lipid recruitment. ATG18a is essential for autophagy under several abiotic stresses, and RNA interference–ATG18a transgenic plants showed an autophagy-defective phenotype during nutrient stress and senescence (35, 36). atg18 mutants show defects in autophagosome formation and display an early senescence phenotype (34). In addition, the ER is a target of autophagy during ER stress in plants, and this ER stress–induced autophagy is dependent on the function of ATG18a (7). Thus, ATG18a is likely to be required for autophagosome formation in Arabidopsis for bulk autophagy and also for reticulophagy during ER stress (7).In this study, we aimed to clarify the role of sulfide in the regulation of autophagy under ER stress through persulfidation of ATG18a. We found that persulfidation affects ATG18a lipid-binding activity, which in turns regulates the number and size of autophagosomes produced upon ER stress.     

Autophagy is required for proper cysteine homeostasis in pancreatic cancer through regulation of SLC7A11

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest forms of cancer and is highly refractory to current therapies. We had previously shown that PDAC can utilize its high levels of basal autophagy to support its metabolism and maintain tumor growth. Consistent with the importance of autophagy in PDAC, autophagy inhibition significantly enhances response of PDAC patients to chemotherapy in two randomized clinical trials. However, the specific metabolite(s) that autophagy provides to support PDAC growth is not yet known. In this study, we demonstrate that under nutrient-replete conditions, loss of autophagy in PDAC leads to a relatively restricted impairment of amino acid pools, with cysteine levels showing a significant drop. Additionally, we made the striking discovery that autophagy is critical for the proper membrane localization of the cystine transporter SLC7A11. Mechanistically, autophagy impairment results in the loss of SLC7A11 on the plasma membrane and increases its localization at the lysosome in an mTORC2-dependent manner. Our results demonstrate a critical link between autophagy and cysteine metabolism and provide mechanistic insights into how targeting autophagy can cause metabolic dysregulation in PDAC.

Despite progress in cancer therapy, the prognosis for pancreatic ductal adenocarcinoma (PDAC) remains extremely poor with a 5-y survival rate of just 9% and it is predicted to become the second leading cause of cancer death in the United States by 2030 (1–3). The PDAC tumor microenvironment is highly desmoplastic and is composed of heterogeneous cell types, as well as an exuberant extracellular matrix. Together, this leads to poor perfusion and extreme hypoxia, creating a nutrient-limited environment with impaired drug penetration (4, 5). Another hallmark of PDAC is elevated basal autophagy which plays multiple protumorigenic roles, including promoting immune evasion and supporting its metabolic demand in this austere microenvironment (6–10). Therefore, clinical strategies have been employed to inhibit autophagy in PDAC patients using lysosomal inhibitors such as chloroquine or hydroxychloroquine (11, 12).While autophagy can support diverse metabolic processes through the degradation of various cargo, how it supports PDAC metabolism has not been fully elucidated. In the present study, we found that autophagy has a selective role in sustaining cysteine (Cys) pools in PDAC. One of the major mechanisms of Cys homeostasis is through the import of cystine (the oxidized dimer of cysteine) through system x_c⁻, a cystine/glutamate antiporter composed of SLC7A11 (xCT) and SLC3A2 (13). Recently, it was shown that both Cys and SLC7A11 are critical for PDAC growth (14). Here, we report that under low Cys conditions, SLC7A11 utilizes autophagy machinery to allow localization at the plasma membrane. Moreover, we demonstrate that loss of autophagy increases phosphorylation of SLC7A11 by mTORC2, and it remains primarily localized at the lysosome where its cystine import function is impaired. In summary, we identify a mechanism of Cys homeostasis in PDAC where the function of SLC7A11 is coordinately sustained by autophagic machinery and mTORC2 activity based on intracellular Cys levels.     

Primary cilia and the reciprocal activation of AKT and SMAD2/3 regulate stretch-induced autophagy in trabecular meshwork cells

Activation of autophagy is one of the responses elicited by high intraocular pressure (IOP) and mechanical stretch in trabecular meshwork (TM) cells. However, the mechanosensor and the molecular mechanisms by which autophagy is induced by mechanical stretch in these or other cell types is largely unknown. Here, we have investigated the mechanosensor and downstream signaling pathway that regulate cyclic mechanical stretch (CMS)-induced autophagy in TM cells. We report that primary cilia act as a mechanosensor for CMS-induced autophagy and identified a cross-regulatory talk between AKT1 and noncanonical SMAD2/3 signaling as critical components of primary cilia-mediated activation of autophagy by mechanical stretch. Furthermore, we demonstrated the physiological significance of our findings in ex vivo perfused eyes. Removal of primary cilia disrupted the homeostatic IOP compensatory response and prevented the increase in LC3-II protein levels in response to elevated pressure challenge, strongly supporting a role of primary cilia-mediated autophagy in regulating IOP homeostasis.

The trabecular meshwork (TM) is a pressure-sensitive tissue located in the anterior segment of the eye and key regulator of intraocular pressure (IOP). Malfunction of this tissue results in improper drainage of aqueous humor outflow, leading to ocular hypertension, the major risk factor for developing glaucoma (1–3). The TM consists of an irregular lattice of collagen beams lined by TM endothelial-like cells, followed by a zone of loose connective tissue containing TM cells, through which aqueous humor must pass before leaving the eye. Changes in pressure gradients and fluid flow associated with eye movement, circadian rhythm, or the ocular pulse cause small and high variations in IOP, which are translated in continuous cycles of tissue deformation and relaxation. Cells in the TM are known to be able to sense these deformations as mechanical forces and respond to them by eliciting a variety of responses, including reorganization of actin cytoskeleton, changes in gene expression, secretion of cytokines, modulation of matrix metalloproteinases, and extracellular-matrix remodeling (reviewed in ref. 4). It is believed that these mechanoresponses are critical regulators of IOP homeostasis; however, the mechanosensors and the downstream mechanotransduction signaling in TM have still not been characterized.Our laboratory has identified the activation of macroautophagy (hereafter autophagy) as one of the responses elicited in TM cells following application of static or cyclic mechanical stretch (CMS) (5–7). Activation of autophagy was also observed quickly after pressure elevation in porcine perfused eyes (5), which prompted us to propose autophagy as a crucial physiological response to adapt to mechanical forces and maintain cellular homeostasis. The exact roles of autophagy in TM cells and tissue function are still under investigation. Most recently, we have shown the CMS-induced translocation of the autophagy marker LC3 (microtubule-associated protein 1 light chain 3) to the nuclear compartment, where it associates with the nucleolus and interacts with the ribophagy receptor NUFIP1 (nuclear FMR1 interacting protein 1), suggesting a potential role of CMS-induced autophagy in surveillance of the nucleolus activity (6). Furthermore, we have also recently provided direct evidence demonstrating autophagy as a regulator of TGF-β/SMAD-induced fibrogenesis in TM cells (8).Autophagy is a fundamental process for degradation or recycling of intracellular components, which is essential to maintain cellular homeostasis. Autophagy occurs constitutively at basal levels, but it is quickly activated upon several types of stress, such as nutrient depletion, pathogen infections, drug treatment, accumulation of aggregated proteins and damaged organelles, and mechanical stress (7, 9–11). The molecular mechanisms by which cells recognize stress and regulate autophagic activity are very complex and differ based on the stimuli. A variety of components, for example, receptor tyrosine kinases, second messengers (Ca²⁺ or cAMP [cyclic adenosine monophosphate]), protein kinases, and downstream autophagy-related (ATG) complexes, participate in the regulation of autophagy (10). Among them, the best characterized is MTOR (mechanistic target of rapamycin kinase), a negative regulator of autophagy (10, 12, 13). Although MTOR acts as a core sensor in autophagic regulation, numerous studies have shown the MTOR-independent autophagy activation upon different stresses (10). Indeed, our own study and that of King et al. showed that the induction of autophagy in response to mechanical stress occurs in an MTOR-independent manner (5, 14). The mechanosensor and the downstream signaling pathway responsible for the activation of autophagy in response to stretching have still not been identified.Primary cilium (PC) is a nonmotile cell-surface projection found almost ubiquitously in vertebrate cells, which acts as a cellular antenna that senses a wide variety of signals, including chemical and mechanical stimuli (15–19). PC plays a critical role in smell, sight, and mechanosensation. PC defects are associated with a number of human diseases termed ciliopathies. The most common feature in patients affected with ciliopathies include visual dysfunction (16). In particular, Lowe syndrome patients often develop ocular hypertension and glaucoma (20). Structurally, the PC consists of a microtubule-based core structure, called axoneme, and a basal body, which is a derivative of the centriole of centrosome from which axoneme is extended and surrounded by ciliary membrane (21). The ciliary membrane is a specialized domain extension of the plasma membrane enriched on signaling receptors and channels, including hedgehog (Hh) and Ca²⁺ pathways, which enables the PC to function as a signaling hub (16, 22, 23). Cargo trafficking into and out of the cilium is mediated by a specialized form of vesicle trafficking, named intraflagellar transport (IFT), that is composed of a multiprotein complex (16, 23).Recent studies have demonstrated the reciprocal relationship between PC and autophagy. Autophagy has been shown to both positively and negatively regulate ciliogenesis. Under nutrient-rich conditions, basal autophagy inhibits cilia growth by limiting trafficking of PC components to the basal body through direct degradation of IFT20 (24). In contrast, nutrient starvation triggers the autophagic degradation of oral-facial-digital syndrome 1 and promotes cilia growth (24, 25). Conversely, functional PC are required for activation of autophagy in response to starvation and fluid flow. In both cases, autophagy was initiated by the recruitment of ATG16L to the basal body, suggesting that this event is a hallmark for PC-induced autophagy. Intriguingly, the signaling pathway mediating PC-induced autophagy activation differed based on the stimuli. While Hh/smoothened (SMO) was reported to mediate activation of autophagy in response to starvation (24), the LKB1–AMPK–MTOR signaling pathway was found to regulate PC-induced autophagy and cell volume in kidney epithelial cells under shear stress (26, 27). Whether PC are also involved in the regulation of autophagy triggered by mechanical stretching has not yet been explored.The purpose of this study is to investigate a potential role of PC in stretch-induced autophagy in TM cells. Here, we report that PC acts as a mechanosensor for CMS-induced autophagy, and we identified AKT1 and SMAD2/3 as critical components of the signal mechanotransduction.     

Yeast optimizes metal utilization based on metabolic network and enzyme kinetics

Metal ions are vital to metabolism, as they can act as cofactors on enzymes and thus modulate individual enzymatic reactions. Although many enzymes have been reported to interact with metal ions, the quantitative relationships between metal ions and metabolism are lacking. Here, we reconstructed a genome-scale metabolic model of the yeast Saccharomyces cerevisiae to account for proteome constraints and enzyme cofactors such as metal ions, named CofactorYeast. The model is able to estimate abundances of metal ions binding on enzymes in cells under various conditions, which are comparable to measured metal ion contents in biomass. In addition, the model predicts distinct metabolic flux distributions in response to reduced levels of various metal ions in the medium. Specifically, the model reproduces changes upon iron deficiency in metabolic and gene expression levels, which could be interpreted by optimization principles (i.e., yeast optimizes iron utilization based on metabolic network and enzyme kinetics rather than preferentially targeting iron to specific enzymes or pathways). At last, we show the potential of using the model for understanding cell factories that harbor heterologous iron-containing enzymes to synthesize high-value compounds such as p-coumaric acid. Overall, the model demonstrates the dependence of enzymes on metal ions and links metal ions to metabolism on a genome scale.

Metabolism plays a key role in all cellular processes. To maintain metabolic activities, cells take up nutrients from the environment, including macronutrients and minerals. Macronutrients such as carbohydrates and amino acids are primary substrates of metabolic reactions to provide energy and precursors for other biological processes. Many minerals (e.g., metal ions) are essential but appear to be less related with metabolism as they do not directly participate in metabolic reactions as either substrates or products. Instead, they serve as cofactors on enzymes to ensure proper function. The BRENDA database (1) summarizes experimental evidence of metal ions being stimulatory or even essential for ion-containing enzyme activities. Although many individual enzymes have been experimentally identified to interact with metal ions (2, 3), there is a lack of quantitative relationships between metal ions and metabolism from a holistic perspective.Predicting cellular behavior with quantitative models is of particular interest (4), which could hopefully aid in uncovering the mechanisms and driving forces by which cells adapt to perturbations (e.g., reduced availability of metal ions). Genome-scale metabolic models (GEMs) together with constraint-based approaches enable predicting the optimal state of cells subject to external and internal constraints based on optimization principles (5, 6). Using GEMs as a framework, many other biological processes than metabolism have been mathematically described, especially protein synthesis processes (7–9). The integration of protein synthesis with metabolism allows for imposing proteome constraints by enzyme kinetics, which has shown wider applications, such as predictions of protein levels (7, 10) and proteome allocation (9, 11). Given that enzyme kinetics could be affected by metal ion availability, it will be possible to apply proteome-constrained frameworks to establish quantitative relationships between metal ions and metabolism.Besides serving as an important cell factory, the yeast Saccharomyces cerevisiae has shown to be a powerful model organism for experimentally investigating metal ions (12, 13) and could also infer targets on metal ion–related diseases in humans based on homology (14). In addition, a wealth of information of metal ions has been accumulated for S. cerevisiae [e.g., a large number of yeast proteins have been identified as iron-containing (15) and zinc-containing (16) proteins]. Therefore, yeast would also be a promising model organism to mathematically explore the effect of metal ions on metabolism.Here, we present a mathematical framework that integrates protein synthesis and incorporation of enzyme cofactors into a GEM of the yeast S. cerevisiae and name the resulting model CofactorYeast. We show the model’s capability of estimating abundances of metal ions binding on enzymes, capturing representative proteins of distinct metal ions, and simulating metabolic responses upon reduced availability of metal ions, especially iron. At last, we illustrate its application in metabolic engineering of yeast for producing para-coumaric acid (also called p-hydroxycinnamic acid [p-HCA]), which could be dependent on optimal iron and proteome allocation.     

Myristoylation alone is sufficient for PKA catalytic subunits to associate with the plasma membrane to regulate neuronal functions

Myristoylation is a posttranslational modification that plays diverse functional roles in many protein species. The myristate moiety is considered insufficient for protein–membrane associations unless additional membrane-affinity motifs, such as a stretch of positively charged residues, are present. Here, we report that the electrically neutral N-terminal fragment of the protein kinase A catalytic subunit (PKA-C), in which myristoylation is the only functional motif, is sufficient for membrane association. This myristoylation can associate a fraction of PKA-C molecules or fluorescent proteins (FPs) to the plasma membrane in neuronal dendrites. The net neutral charge of the PKA-C N terminus is evolutionally conserved, even though its membrane affinity can be readily tuned by changing charges near the myristoylation site. The observed membrane association, while moderate, is sufficient to concentrate PKA activity at the membrane by nearly 20-fold and is required for PKA regulation of AMPA receptors at neuronal synapses. Our results indicate that myristoylation may be sufficient to drive functionally significant membrane association in the absence of canonical assisting motifs. This provides a revised conceptual base for the understanding of how myristoylation regulates protein functions.

Myristoylation is a major type of posttranslational modification that occurs at the N terminus of a myriad of proteins (1–4). Depending on the target, myristoylation can contribute to the structure, stability, protein–protein interactions, and subcellular localization of the modified proteins (2–4). In particular, myristoylation often facilitates protein association with the membrane. However, it is thought that, with an acyl chain of only 14 carbons, myristate confers insufficient energy for stable association of a protein with the membrane (5, 6). Subsequent studies have shown that a second membrane-affinity motif, such as a stretch of basic residues or a second lipid modification, is required for the membrane association of several myristoylated proteins (reviewed in refs. 2–4). When the second membrane-affinity motif is removed or neutralized, either physiologically or via mutagenesis, the membrane localization of the protein is disrupted. Thus, the canonical view is that myristoylation alone is not sufficient to provide a functionally significant association of a protein with the plasma membrane, even though myristoylation has been observed to be associated with reconstituted lipid bilayers (7).Myristoylation was first discovered in the catalytic subunit of protein kinase A (PKA) (1, 8), which is a primary mediator of the second messenger cAMP that plays diverse essential roles in nearly all organisms, from bacteria to humans. At rest, PKA is a tetrameric protein that consists of two regulatory subunits (PKA-Rs) and two catalytic subunits (PKA-Cs). PKA holoenzymes are anchored to specific subcellular locations via the binding of PKA-R with A-Kinase anchoring proteins (9–12). In the presence of cAMP, PKA-C is released from PKA-R and becomes an active kinase (8, 13–16).Despite being myristoylated, PKA-C is thought to function as a cytosolic protein because of its high solubility (14, 15). Consistently, a PKA-C mutant with disrupted myristoylation has been shown to support the phosphorylation of certain substrates and to maintain several PKA functions in heterologous cells (17). Structural studies found that the PKA-C myristoylation is folded into a hydrophobic pocket, and it was proposed that this myristoylation serves a structural role (18–20). This view has started to shift based on recent reports showing that activated PKA-C can associate with the membrane in a myristoylation-dependent manner (16, 21, 22), including in neurons. However, the extent to which PKA-C associates with the plasma membrane in living cells and its functional significance are not known. Furthermore, as discussed above, myristolyation-mediated membrane association is thought to require a second-membrane motif. The identity of this second membrane-affinity motif has not been determined. Therefore, we set out to address these questions.     

Mitochondrial metabolism is essential for invariant natural killer T cell development and function

Conventional T cell fate and function are determined by coordination between cellular signaling and mitochondrial metabolism. Invariant natural killer T (iNKT) cells are an important subset of “innate-like” T cells that exist in a preactivated effector state, and their dependence on mitochondrial metabolism has not been previously defined genetically or in vivo. Here, we show that mature iNKT cells have reduced mitochondrial respiratory reserve and iNKT cell development was highly sensitive to perturbation of mitochondrial function. Mice with T cell-specific ablation of Rieske iron-sulfur protein (RISP; T-Uqcrfs1^−/−), an essential subunit of mitochondrial complex III, had a dramatic reduction of iNKT cells in the thymus and periphery, but no significant perturbation on the development of conventional T cells. The impaired development observed in T-Uqcrfs1^−/− mice stems from a cell-autonomous defect in iNKT cells, resulting in a differentiation block at the early stages of iNKT cell development. Residual iNKT cells in T-Uqcrfs1^−/− mice displayed increased apoptosis but retained the ability to proliferate in vivo, suggesting that their bioenergetic and biosynthetic demands were not compromised. However, they exhibited reduced expression of activation markers, decreased T cell receptor (TCR) signaling and impaired responses to TCR and interleukin-15 stimulation. Furthermore, knocking down RISP in mature iNKT cells diminished their cytokine production, correlating with reduced NFATc2 activity. Collectively, our data provide evidence for a critical role of mitochondrial metabolism in iNKT cell development and activation outside of its traditional role in supporting cellular bioenergetic demands.

Cellular metabolic pathways are interwoven with traditional signaling pathways to regulate the function and differentiation of T cells (1–3). Upon activation, effector T cells display a marked increase in glycolytic metabolism even in the presence of ample oxygen, termed aerobic glycolysis (4). We have previously shown that despite increased aerobic glycolysis, T cell activation depends on mitochondrial metabolism for generation of reactive oxygen species (ROS) for signaling (5). As activated T cells progress to a memory or regulatory phenotype, they preferentially oxidize fatty acids to support mitochondrial metabolism, and enhanced fatty acid oxidation (FAO) and spare respiratory capacity (SRC) are essential to maintenance of their phenotype (6, 7).CD1d-restricted invariant natural killer T (iNKT) cells are a unique subset of lymphocytes that exhibit a preactivated phenotype with rapid effector responses (8, 9). iNKT cells are capable of producing large amount of proinflammatory and antiinflammatory cytokines thus have broad immunomodulatory roles (8–10). Given that these cells are poised for rapid proliferation and cytokine production, we hypothesized that coordination of cellular signaling with cellular metabolism will be especially critical for optimal iNKT function. In support of this hypothesis, several studies suggest that modulation of cellular metabolism affects iNKT cell development and function. iNKT cell development is diminished upon deletion of the miR-181 a1b1 cluster, which regulates phosphoinositide 3-kinase signaling and decreases aerobic glycolysis (11, 12). In addition, T cell-specific deletion of Raptor (a component of mTORC1), a metabolic regulator, leads to defects in iNKT cell development and function (13, 14). Loss of folliculin-interacting protein 1 (Fnip1), an adaptor protein that physically interacts with AMP-activated protein kinase, also results in defective NKT cell development, and interestingly conventional T cells develop normally (15). Furthermore, a number of studies targeting bioenergetics processes or related molecules, like alteration of glucose metabolism, mitochondrial-targeted antioxidant treatment, and receptor-interacting protein kinase 3-dependent activation of mitochondrial phosphatase, showed significant effects on iNKT cell ratio and function (16–19). A recent study showed that iNKT cells are less efficient in glucose uptake than CD4⁺ T cells. Furthermore, activated iNKT cells preferentially metabolize glucose by the pentose phosphate pathway and mitochondria, instead of converting into lactate, since high lactate environment is detrimental to their homeostasis and effector function (20).In conventional lymphocytes, mitochondria clearly play a role in coordination of cell signaling and cell fate decisions outside of production of energy (5, 21–23). During T cell activation mitochondria localize at immune synapses that T cells form with antigen-presenting cells (22). T cell receptor (TCR) stimulation triggers mitochondrial ROS (mROS) production as well as mitochondrial ATP production that are released at the immune synapses and are critical for Ca²⁺ homeostasis and modulation of TCR-induced downstream signaling pathways (22). We previously showed that mice with T-cell–specific deletion of Rieske iron sulfur protein (RISP), a component of mitochondrial complex III of the mitochondrial electron transport chain (ETC), are defective in antigen-specific T cell activation due to deficiency of mROS required for cellular signaling (5). Several recent studies showed that ROS or factors that affect ROS production are also important in iNKT cell development and effector functions (24–27). In addition, inhibition of mitochondrial oxidative phosphorylation (OXPHOS) by oligomycin has been shown to decreased survival and cytokine production by splenic iNKT cells (20). However, the requirement of mitochondrial metabolism for iNKT cell development and function has not been previously defined genetically or in vivo.Here we showed that iNKT cells have comparable basal mitochondrial oxygen consumption to conventional T cells but displayed lower SRC and FAO, which are thought to impart cells with mitochondrial reserve under stress. Using Uqcrfs1^fl/fl;CD4-Cre⁺ (hereafter referred as T-Uqcrfs1^−/−) mice, we showed that abrogation of mitochondrial metabolism resulted in a cell-autonomous defect in iNKT cell development in thymus and periphery. The iNKT cells were able to proliferate but exhibited impaired activation, suggesting that they were not lacking bioenergetically but rather had aberrant TCR signaling in vivo, leading to altered expression of downstream factors required for their terminal maturation. Accordingly, T-Uqcrfs1^−/− iNKT cells displayed lower T-bet and CD122 levels and did not respond to interleukin (IL)-15 stimulation. Knockdown of RISP in mature iNKT cells also limited NFATc2 translocation to the nucleus. Collectively, our data highlighted an important role of mitochondrial metabolism in modulating TCR signaling in vivo and regulating iNKT cell development and function.     

Snx14 proximity labeling reveals a role in saturated fatty acid metabolism and ER homeostasis defective in SCAR20 disease

Fatty acids (FAs) are central cellular metabolites that contribute to lipid synthesis, and can be stored or harvested for metabolic energy. Dysregulation in FA processing and storage causes toxic FA accumulation or altered membrane compositions and contributes to metabolic and neurological disorders. Saturated lipids are particularly detrimental to cells, but how lipid saturation levels are maintained remains poorly understood. Here, we identify the cerebellar ataxia spinocerebellar ataxia, autosomal recessive 20 (SCAR20)-associated protein Snx14, an endoplasmic reticulum (ER)–lipid droplet (LD) tethering protein, as a factor required to maintain the lipid saturation balance of cell membranes. We show that following saturated FA (SFA) treatment, the ER integrity of SNX14^KO cells is compromised, and both SNX14^KO cells and SCAR20 disease patient-derived cells are hypersensitive to SFA-mediated lipotoxic cell death. Using APEX2-based proximity labeling, we reveal the protein composition of Snx14-associated ER–LD contacts and define a functional interaction between Snx14 and Δ-9 FA desaturase SCD1. Lipidomic profiling reveals that SNX14^KO cells increase membrane lipid saturation following exposure to palmitate, phenocopying cells with perturbed SCD1 activity. In line with this, SNX14^KO cells manifest delayed FA processing and lipotoxicity, which can be rescued by SCD1 overexpression. Altogether, these mechanistic insights reveal a role for Snx14 in FA and ER homeostasis, defects in which may underlie the neuropathology of SCAR20.

Cells regularly internalize exogenous fatty acids (FAs) and must remodel their metabolic pathways to process and properly store FA loads. As it is a central cellular currency that can be stored, incorporated into membrane lipids, or harvested for energy, cells must balance FA uptake, processing, and oxidation to maintain homeostasis. Defects in any of these elevates intracellular free FAs (FFAs), which can act as detergents and damage organelles. Excessive membrane lipid saturation can also alter organelle function and contribute to cellular pathology, known as lipotoxicity (1, 2). Failure to properly maintain lipid compositions and storage contributes to many metabolic disorders (3), including type 2 diabetes (4), obesity (5), cardiac failure (6, 7), and various neurological diseases (8).Properties of FAs such as their degree of saturation and chain length are key determinants of their fate within the cell (9). High concentrations of saturated FAs (SFAs) in particular are highly toxic, as their incorporation into organelles affects membrane fluidity and can trigger lipotoxicity and cell death (10–13). To prevent this, cells desaturate SFAs into monounsaturated FAs (MUFAs) before they are subsequently incorporated into membrane glycerophospholipids or stored as triglycerides (TGs) in lipid droplets (LDs). LD production provides a lipid reservoir to sequester otherwise toxic FAs, providing a metabolic buffer to maintain lipid homeostasis (14, 15).As LDs are created by and emerge from the ER network, interorganelle communication between the ER and LDs is vital for LD biogenesis (16). Consequently, numerous proteins that contribute to LD biogenesis, such as seipin (17, 18) and the diacylglyceride acyltransferase (DGAT) (19), are implicated in ER–LD cross-talk. Previously, we identified Snx14, a sorting nexin (SNX) protein linked to the cerebellar ataxia disease spinocerebellar ataxia, autosomal recessive 20 (SCAR20) (20–22), as a novel factor that promotes FA-stimulated LD growth at ER–LD contacts (23, 24). Snx14 is an ER-anchored integral membrane protein. During periods of elevated FA flux, Snx14 is recruited to ER–LD contact sites, where it promotes the incorporation of FAs into TG as LDs grow (23). In line with this, SNX14^KO cells exhibit defective LD morphology following oleate addition, implying Snx14 is required for proper FA storage in LDs. Related studies of Snx14 homologs in yeast and Drosophila indicate a conserved role for Snx14-family proteins in FA homeostasis and LD biogenesis (25, 26).Despite these insights, why humans with Snx14 loss-of-function mutations develop the cerebellar ataxia disease SCAR20 remains enigmatic. Given the proposed role of Snx14 in lipid metabolism, and that numerous neurological pathologies arise through defects in ER lipid homeostasis (27–29), here we investigated whether Snx14 loss alters the ability of cells to maintain lipid homeostasis in response to FA influx. Our findings indicate that Snx14-deficient cells are hypersensitive to SFA exposure and manifest defects in ER morphology and ER-associated lipid metabolism.     

Antipsychotic drugs counteract autophagy and mitophagy in multiple sclerosis

Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease characterized by myelin damage followed by axonal and ultimately neuronal loss. The etiology and physiopathology of MS are still elusive, and no fully effective therapy is yet available. We investigated the role in MS of autophagy (physiologically, a controlled intracellular pathway regulating the degradation of cellular components) and of mitophagy (a specific form of autophagy that removes dysfunctional mitochondria). We found that the levels of autophagy and mitophagy markers are significantly increased in the biofluids of MS patients during the active phase of the disease, indicating activation of these processes. In keeping with this idea, in vitro and in vivo MS models (induced by proinflammatory cytokines, lysolecithin, and cuprizone) are associated with strongly impaired mitochondrial activity, inducing a lactic acid metabolism and prompting an increase in the autophagic flux and in mitophagy. Multiple structurally and mechanistically unrelated inhibitors of autophagy improved myelin production and normalized axonal myelination, and two such inhibitors, the widely used antipsychotic drugs haloperidol and clozapine, also significantly improved cuprizone-induced motor impairment. These data suggest that autophagy has a causal role in MS; its inhibition strongly attenuates behavioral signs in an experimental model of the disease. Therefore, haloperidol and clozapine may represent additional therapeutic tools against MS.

Multiple sclerosis (MS) is a neuroinflammatory disease, characterized by progressive neurological damage consisting in crumbling of the axonal myelin sheath in the central nervous system (CNS), followed by strong impairment in axonal conductance, axonal transection, and death of oligodendrocytes and neurons (1). MS onset is subacute or chronic, typically in a relapsing–remitting fashion, with phases of neuroinflammation corresponding to clinically evident disease followed by periods of oligodendrocyte proliferation, partial reconstitution of the myelin sheath, and corresponding clinical recovery. MS worsens over time, evolving into a progressive form, characterized by the disappearance of the relapsing–remitting phases and by permanent chronic lesions.Inflammation plays a central and fundamental role in MS (2). The initial phase of MS is characterized by signs of an autoimmune response including disruption of the blood–brain barrier, invasion of the CNS by immune cells, and presence of antibodies against myelin. These are believed to play a causative role in the onset of the disease. Immunological aggression is sustained by a macrophage- or microglia-driven secretion of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) (3, 4) or interleukin-1 beta (IL-1-β) (5, 6), which have been shown to promote neuronal, oligodendrocyte, and vascular damage in models of MS.Autophagy may also play a fundamental pathophysiological role in MS, but this has been only marginally explored thus far (1, 7). Autophagy represents a key intracellular function, which allows the cell to face periods of nutrient deprivation, meanwhile eliminating damaged and potentially harmful molecules, organelles, or invading microorganisms (8). These activities are grounded on the capacity of the cell to organize a double-membrane–lined autophagosome that, after engulfing the unwanted material, brings it to the lysosomal compartment, which in turn ensures degradation of proteins and recycling of nutrients.Many neurodegenerative diseases are characterized by pathological accumulation of misfolded or damaged proteins, suggesting an important role of autophagy in their pathophysiology (9, 10). In Alzheimer’s disease, for example, impairment of the autophagic flux due to reduced vesicle clearance has been associated to accumulation of an aggregated form of hyperphosphorylated tau protein and to extracellular amyloid-β (A-β) plaques (11). Similarly, in Parkinson’s disease, dysfunctional lysosomes and accumulation of autophagosomes are associated with intracellular inclusions of α-synuclein and other polyubiquitinated proteins.Several reports have demonstrated an increase in autophagic markers in human samples of MS patients (12). In particular, our and other groups have shown that autophagic and mitophagic markers are increased in serum and cerebrospinal fluid (CSF) of MS-affected individuals (13–15). However, the role of autophagy in MS remains still controversial. (16, 17).In this study, we investigated in detail the involvement of autophagy in MS. We used in vitro, ex vivo, and in vivo models to test the effect of proinflammatory cytokines, lysolecithin (LPC), and cuprizone (CPZ) on myelin production, axonal myelination, and motor performance. In particular, we investigated the occurrence of autophagy in the different models and examined the effect of autophagy inhibitors and of two antipsychotic drugs, clozapine and haloperidol, on autophagy and behavior, revealing a significant improvement in motor function in MS animals.The rationale for employing these antipsychotic drugs stems from neuroimaging and postmortem investigations on the brain of schizophrenia patients that revealed loss of white matter (18, 19) and down-regulation of genes involved in oligodendrocyte functioning and myelination (20, 21). Antipsychotic drugs, in particular haloperidol and clozapine, proved effective in improving cortical myelination as well as spatial working memory and locomotor activity in animal models (22–25). The molecular mechanism(s) underlying these effects are still obscure, but it has been recently suggested that these antipsychotic drugs may interfere with the autophagic process (26, 27). Our findings explore this mechanism of action of haloperidol and clozapine and disclose the opportunity of repurposing these drugs that are currently employed in other clinical settings for the treatment of MS.     

Cell dispersal by localized degradation of a chemoattractant

Chemotaxis, the guided motion of cells by chemical gradients, plays a crucial role in many biological processes. In the social amoeba Dictyostelium discoideum, chemotaxis is critical for the formation of cell aggregates during starvation. The cells in these aggregates generate a pulse of the chemoattractant, cyclic adenosine 3’,5’-monophosphate (cAMP), every 6 min to 10 min, resulting in surrounding cells moving toward the aggregate. In addition to periodic pulses of cAMP, the cells also secrete phosphodiesterase (PDE), which degrades cAMP and prevents the accumulation of the chemoattractant. Here we show that small aggregates of Dictyostelium can disperse, with cells moving away from instead of toward the aggregate. This surprising behavior often exhibited oscillatory cycles of motion toward and away from the aggregate. Furthermore, the onset of outward cell motion was associated with a doubling of the cAMP signaling period. Computational modeling suggests that this dispersal arises from a competition between secreted cAMP and PDE, creating a cAMP gradient that is directed away from the aggregate, resulting in outward cell motion. The model was able to predict the effect of PDE inhibition as well as global addition of exogenous PDE, and these predictions were subsequently verified in experiments. These results suggest that localized degradation of a chemoattractant is a mechanism for morphogenesis.

Eukaryotic cell motion guided by gradients of diffusible chemoattractants plays a crucial role in wound healing, embryology, the movement of immune cells, and cancer metastasis (1–4). In some cell types, including neutrophils and the social amoeba Dictyostelium discoideum, gradient generation occurs through a relay mechanism where cells stimulated by the chemoattractant secrete additional chemoattractant (5–7). To prevent continuous build-up of chemoattractants, many systems use enzymes to degrade the chemoattractant. These enzymes are either membrane-bound, with their active sites facing the extracellular environment, or can diffuse in the surrounding environment (8, 9). In addition, chemoattractants may be removed by other methods, including receptor uptake or decoy receptors (10, 11).Compared to chemoattraction, relatively little is known about chemorepulsion, exemplified by the dispersal of immune cells out of a tissue during the resolution of inflammation. Under some conditions in chamber-based assays, chemoattractant-degrading enzymes can create a local minimum in chemoattractant concentration, resulting in chemoattractant gradients that point away from areas with high cell density (12–14).In a nutrient-rich environment, Dictyostelium cells grow as separate, independent cells. When deprived of food, these amoebae start to secrete the chemoattractant cyclic adenosine 3′,5′-monophosphate (cAMP) in an oscillatory manner (15, 16). The secreted cAMP rapidly diffuses to neighboring cells, which, in turn, start to secrete cAMP as well. The resulting relay mechanism generates periodic waves that sweep through the cell population with a period of 6 min to 10 min (17). For wave periods smaller than 10 min, cells only respond to incoming waves, ignoring the “back of the wave,” and directionally move toward higher concentrations of cAMP (18, 19), form streams, and eventually aggregate into mounds of up to ∼100,000 cells. Cells within the aggregate subsequently differentiate and form a fruiting body that contains the majority of the original population of cells as a mass of spores held up off of the substrate by a stalk to maximize dispersal of spores (15, 16). In addition to cAMP, Dictyostelium cells also secrete phosphodiesterases (PDEs), which hydrolyze cAMP and which prevent continuous build-up of cAMP (8, 20). The presence of PDE is essential for aggregation, and mutants that cannot secrete PDE fail to form viable aggregation centers (21).We reasoned that the competition between a time-varying chemoattractant signal and an inhibitor can result in guidance that changes direction as a function of time. To test this hypothesis, we examined the aggregation of Dictyostelium cells at low cell densities. We present evidence that aggregates of developing Dictyostelium cells display dispersal behavior in which cells are “repelled” from, rather than attracted to, aggregates. This behavior was only present for small aggregates. Furthermore, we show that, during this dispersal behavior, oscillatory cAMP signaling is still active, but that its period is abruptly increased at the onset of dispersal. We develop a model for cell aggregation and show that periodic signaling of cAMP, together with a spatial profile of PDE, can explain the observed dispersal. Furthermore, the model predicts that the disruption of the PDE profile, either by removal of PDE or by globally adding PDE, will result in the abolishment of dispersal. These predictions were subsequently verified in experiments. Our results suggest that, by modulating the frequency of cAMP signaling, small aggregates can shed their cells, potentially avoiding mounds that would form small and thus relatively ineffective fruiting bodies.     

Spatio-temporal heterogeneity in hippocampal metabolism in control and epilepsy conditions

The hippocampus’s dorsal and ventral parts are involved in different operative circuits, the functions of which vary in time during the night and day cycle. These functions are altered in epilepsy. Since energy production is tailored to function, we hypothesized that energy production would be space- and time-dependent in the hippocampus and that such an organizing principle would be modified in epilepsy. Using metabolic imaging and metabolite sensing ex vivo, we show that the ventral hippocampus favors aerobic glycolysis over oxidative phosphorylation as compared to the dorsal part in the morning in control mice. In the afternoon, aerobic glycolysis is decreased and oxidative phosphorylation increased. In the dorsal hippocampus, the metabolic activity varies less between these two times but is weaker than in the ventral. Thus, the energy metabolism is different along the dorsoventral axis and changes as a function of time in control mice. In an experimental model of epilepsy, we find a large alteration of such spatiotemporal organization. In addition to a general hypometabolic state, the dorsoventral difference disappears in the morning, when seizure probability is low. In the afternoon, when seizure probability is high, the aerobic glycolysis is enhanced in both parts, the increase being stronger in the ventral area. We suggest that energy metabolism is tailored to the functions performed by brain networks, which vary over time. In pathological conditions, the alterations of these general rules may contribute to network dysfunctions.

Energy production in brain cells is assumed to be optimized to perform tasks or activities in a brain region–specific manner (1–3). Simultaneously, neuronal activity should be adapted to minimize the energy expenditure required for its fueling (4, 5). Several metabolic pathways are available for energy production, in particular glycolysis and oxidative phosphorylation (1). Whether specific metabolic pathways are preferably recruited in different brain areas in a task-dependent manner is not known. The hippocampus is an ideal region to test this hypothesis. The dorsal hippocampus (DH) is involved in learning and memory associated with navigation, exploration, and locomotion, whereas the ventral hippocampus (VH) is involved in motivational and emotional behavior (6–8). These functions are supported by very distinct anatomical (9, 10), morphological (11–13), molecular (14–19), and electrophysiological (12, 13, 20–23) properties of hippocampal cells. The hippocampus structure is also highly heterogeneous at the gene level, from its dorsal to its ventral tip (24, 25), which may serve as a substrate for different functional networks related to cognition and emotion to emerge (7, 26, 27). Given the hippocampus’s heterogeneity, from structure to function, along its dorsoventral axis, our first hypothesis is that energy production is different between the VH and the DH.If energy production is tailored to a given structure–function relationship, we predicted that a change in energy production should accompany a change in the hippocampus’s functional state. Epilepsy is a particularly relevant situation to test this hypothesis. The different types of epilepsies are associated with numerous metabolic and bioenergetic alterations (28). Hypometabolism of epileptic regions is a common signature of mesial temporal lobe epilepsy (TLE) in humans and animal models, such as the pilocarpine model (29, 30). Importantly, in patients with TLE, only the temporal part (including the hippocampus) is epileptogenic. The temporal part corresponds to the ventral part in rodents, which has been identified as the epileptogenic region in the pilocarpine mode (31). Our second hypothesis is that any dorsoventral organization of energy metabolism found in control condition is altered in epilepsy.Finally, hippocampal functions demonstrate circadian regulation (32), in particular place cell properties (33) and long-term synaptic plasticity (34) as well as memory and learning processes (35, 36). Our third hypothesis is that energy production, specifically the respective contributions of oxidative phosphorylation and aerobic glycolysis (3), vary as a function of the time of the day in control and epilepsy.To test these three hypotheses, we used an ex vivo approach to evoke an energy-demanding electrophysiological activity standardized for the three independent variables considered here: time, region (DH/VH), and perturbation (control/epilepsy). As a first step toward a better understanding of the time regulation of hippocampal metabolism, we considered two time points during the night/day cycle: Zeitgeber 3 (ZT3) and Zeitgeber 8 (ZT8), as they correspond to low and high seizure probability in the TLE model used (37). We found that the control DH and VH have distinct time-dependent metabolic signatures regarding glycolysis and oxidative phosphorylation. In experimental epilepsy, there is no more dissociation between DH and VH at ZT3, but the regional difference reappears at ZT8.     

CAP1 binds and activates adenylyl cyclase in mammalian cells

CAP1 (Cyclase-Associated Protein 1) is highly conserved in evolution. Originally identified in yeast as a bifunctional protein involved in Ras-adenylyl cyclase and F-actin dynamics regulation, the adenylyl cyclase component seems to be lost in mammalian cells. Prompted by our recent identification of the Ras-like small GTPase Rap1 as a GTP-independent but geranylgeranyl-specific partner for CAP1, we hypothesized that CAP1-Rap1, similar to CAP-Ras-cyclase in yeast, might play a critical role in cAMP dynamics in mammalian cells. In this study, we report that CAP1 binds and activates mammalian adenylyl cyclase in vitro, modulates cAMP in live cells in a Rap1-dependent manner, and affects cAMP-dependent proliferation. Utilizing deletion and mutagenesis approaches, we mapped the interaction of CAP1-cyclase with CAP’s N-terminal domain involving critical leucine residues in the conserved RLE motifs and adenylyl cyclase’s conserved catalytic loops (e.g., C1a and/or C2a). When combined with a FRET-based cAMP sensor, CAP1 overexpression–knockdown strategies, and the use of constitutively active and negative regulators of Rap1, our studies highlight a critical role for CAP1-Rap1 in adenylyl cyclase regulation in live cells. Similarly, we show that CAP1 modulation significantly affected cAMP-mediated proliferation in an RLE motif–dependent manner. The combined study indicates that CAP1-cyclase-Rap1 represents a regulatory unit in cAMP dynamics and biology. Since Rap1 is an established downstream effector of cAMP, we advance the hypothesis that CAP1-cyclase-Rap1 represents a positive feedback loop that might be involved in cAMP microdomain establishment and localized signaling.

CAP/srv2 was originally identified in yeast biochemically as an adenylyl cyclase–associated protein (1) and genetically as a suppressor of the hyperactive Ras2-V19 allele (2). CAP/srv2-deficient yeast cells are unresponsive to active Ras2, and adenylyl cyclase activity is no longer regulated by Ras2 in these cells (1, 2), indicating the involvement of CAP/srv2 in the Ras/cyclase pathway. However, some mutant CAP/srv2 alleles presented phenotypes not observed in strains with impaired Ras/cyclase pathway (1–3), indicating the existence of Ras/cyclase-independent functions downstream of CAP/srv2. These two phenotype groups, that is, Ras/cyclase-linked and Ras/cyclase-independent, could be suppressed by expression of an N-terminal half and a C-terminal half of CAP/srv2, respectively (4). Subsequent studies showed that the C-terminal half of CAP/srv2 was able to bind monomeric G-actin (5–8) and other actin regulators establishing a role in F-actin dynamics (9–16). Thus, CAP/srv2 is a bifunctional protein with an N-terminal domain involved in Ras/cyclase regulation and a C-terminal domain involved with F-actin dynamics regulation (16–18).CAP1 is structurally conserved in all eukaryotes (18–22); however, their functions are not. Expression of the closely related Schizosaccharomyces pombe cap or mammalian CAP1 in yeast can only suppress the phenotypes associated with deletion of CAP/srv2’s C-terminal but not its N-terminal domain (19, 20, 22), suggesting that only the F-actin dynamics function was conserved while the Ras/cyclase regulation diverged early on in evolution (16–18). CAP/srv2’s N-terminal 1 to 36 domain was sufficient for cyclase binding in yeast involving a conserved RLE motif with predicted coiled-coil folding (23). Interestingly, this domain is also involved in CAP1 oligomerization both in yeast and mammalian cells (24–26), where it purifies as a high-molecular complex of ∼600 kDa consistent with a 1:1 stoichiometric CAP1-actin hexameric organization (12, 25, 27, 28). Importantly, removal of this domain disrupted CAP1 oligomerization, reduced F-actin turnover in vitro and caused defects in cell growth, cell morphology, and F-actin organization in vivo (24, 29). However, whether the conserved RLE motif in mammalian CAP1 interacts with other coiled-coil–containing proteins is for the moment unknown.Ras2-mediated cyclase regulation in yeast requires its farnesylation (30–32). However, the lipid target involved was not identified in the original studies. We have recently shown that mammalian CAP1 interacts with the small GTPase Rap1. The interaction involves Rap1’s C-terminal hypervariable region (HVR) and its lipid moiety in a geranylgeranyl-specific manner; that is, neither the closely related Ras1 nor engineered farnesylated Rap1 interacted with CAP1 (33). Thus, we raised the question whether CAP1-Rap1, similar to CAP/srv2-Ras2 in yeast, plays a role in cAMP dynamics in mammalian cells.In this study, we report that CAP1 binds to and activates mammalian adenylyl cyclase in vitro. The interaction involves CAP1’s conserved RLE motifs and cyclase’s conserved catalytic subdomains (e.g., C1a and/or C2a). Most importantly, we show that both CAP1 and Rap1 modulate cAMP dynamics in live cells and are critical players in cAMP-dependent proliferation.     

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.     

Single-cell visualization and quantification of trace metals in Chlamydomonas lysosome-related organelles

The acidocalcisome is an acidic organelle in the cytosol of eukaryotes, defined by its low pH and high calcium and polyphosphate content. It is visualized as an electron-dense object by transmission electron microscopy (TEM) or described with mass spectrometry (MS)–based imaging techniques or multimodal X-ray fluorescence microscopy (XFM) based on its unique elemental composition. Compared with MS-based imaging techniques, XFM offers the additional advantage of absolute quantification of trace metal content, since sectioning of the cell is not required and metabolic states can be preserved rapidly by either vitrification or chemical fixation. We employed XFM in Chlamydomonas reinhardtii to determine single-cell and organelle trace metal quotas within algal cells in situations of trace metal overaccumulation (Fe and Cu). We found up to 70% of the cellular Cu and 80% of Fe sequestered in acidocalcisomes in these conditions and identified two distinct populations of acidocalcisomes, defined by their unique trace elemental makeup. We utilized the vtc1 mutant, defective in polyphosphate synthesis and failing to accumulate Ca, to show that Fe sequestration is not dependent on either. Finally, quantitation of the Fe and Cu contents of individual cells and compartments via XFM, over a range of cellular metal quotas created by nutritional and genetic perturbations, indicated excellent correlation with bulk data from corresponding cell cultures, establishing a framework to distinguish the nutritional status of single cells.

Trace metals like iron (Fe), copper (Cu), manganese (Mn), and zinc (Zn) play a crucial role as cofactors, providing a range of chemical capabilities to enzymes central to life. Metals also provide structural stability to proteins (1, 2) and enable the catalysis of essential metabolic reactions by providing functional groups that are not readily available via the side chains of amino acids. Consequently, trace metals are required in ∼40% of all enzymes as part of their catalytic centers (3, 4). Individual enzymes are often optimized to utilize a specific metal cofactor, with respect to the chemical utility of that particular trace metal for the catalyzed reaction, the metal’s specific requirements for the binding site in the protein (dimensions, charge, coordination preferences), and the availability of the trace metal within the organism’s reach. There is, however, the possibility of mismetalation, largely attributed to protein structural flexibility and somewhat similar ionic radii and coordination preferences of first-row trace metals (5, 6). Enzyme mismetalation can harm the cell directly by loss of function (7, 8), by accumulation of unintended products, or by the production of toxic side products, for example, reactive oxygen species (9). Cells have therefore developed elaborate strategies to facilitate correct metalation, including preassembling metal cofactors (which allows for easier distinction and delivery of specific metals), the use of metallochaperones (removing especially thermodynamically favored elements like Cu/Zn from the accessible, intracellular trace metal pool), and the compartmentalization of trace metal metabolism (adjusting metal concentrations locally in order to direct binding to target proteins) (6, 10, 11).Chlamydomonas reinhardtii is a unicellular green alga that has been widely used as a eukaryotic, photosynthetic reference system, and therefore exploited in our laboratory to study trace metal metabolism. It has a short generation time (∼6 h), is a facultative heterotroph, and can be grown to high densities (12). Chlamydomonas requires a broad spectrum of metal cofactors to sustain its photosynthetic, respiratory, and metabolic capabilities, with Fe, Cu, Mn, and Zn as the major first-row trace metals involved in these processes. In the last 20 y, studies have revealed a repertoire of assimilatory and distributive transporters in Chlamydomonas, using biochemical and genomics approaches (13–17), discovered mechanisms for metal sparing and recycling to ensure economy (18–21), and identified metal storage sites (2, 22–24).Storage sites are crucial components of trace metal homeostasis. The capacity to sequester individual trace metals is important for controlling protein metalation, detoxification in situations of overload, and buffering during metabolic remodeling. Metal storage provides a selective advantage in competitive environments when transition metals are scarce. One well-known storage site is ferritin, a soluble, mostly cytosolic (animals) or mostly plastidic (plants), 24-subunit oligomer that can oxidize ferrous to ferric Fe and store up to ∼4,500 ferric ions in mineralized form in its core (25, 26). The importance of ferritin as an Fe store is well documented in eukaryotes. In Chlamydomonas, the pattern of expression of ferritin is more consistent with a role in buffering Fe during metabolic transitions, for example from phototrophy to heterotrophy during Fe starvation (23, 27). Vacuoles, lysosome-related, and other acidic organelles, are equally important storage organelles in eukaryotes (28–33). In yeast and plants, these organelles can sequester metals for future use (34). Chlorophyte algae employ a set of smaller cytosolic vacuoles, including contractile vacuoles and acidocalcisomes. Contractile vacuoles (CVs) manage the water content in the cytoplasm; in a fresh water alga that is predominantly facing hypotonic environments (35, 36), water is removed from the cell, potentially using potassium (K) and/or chloride (Cl) (37) to generate an osmotic gradient to attract water to the CV. Acidocalcisomes are lysosome-related organelles in the cytosol, defined by their low pH and high levels of calcium and polyphosphate (polyP) content (38, 39). In Chlamydomonas and other green algae this organelle may also contain K (40, 41). Acidocalcisomes can be identified as electron-dense granules by transmission electron microscopy (TEM) or by utilizing specific probes targeting either the low pH environment or the specific elemental makeup of the compartment (24, 42–44). In addition, acidocalcisomes have been visualized using their unique elemental signature by mass spectrometry (MS)-based imaging techniques, specifically nanoscale secondary ion mass spectrometry (nanoSIMS), or X-ray fluorescence microscopy (XFM) (45, 46). These methods demonstrated that, at least in Chlamydomonas, the acidocalcisome can house high amounts of Cu and Mn in excess conditions (24, 47), making it a prime candidate for Fe storage as well.XFM is a synchrotron-based technique that utilizes X-rays to produce photons in the object to be visualized. The energies of these fluorescent photons are element specific and can be used to determine elemental distribution and concentrations in whole cells or subcellular compartments (48–50). High energy X-rays (>10 keV) penetrate biological material deep enough so that no sectioning is required. True elemental distributions are obtained when metabolic states are preserved rapidly using either vitrification or chemical fixation. XFM has previously been utilized to determine the elemental content of vacuoles of phagocytes infected with various Mycobacterium species (51), to demonstrate transient zinc relocation to the nucleus during macrophage differentiation (52) and the mobilization of Cu during angiogenesis (53).In this work, we employed XFM with sub-100-nm spatial resolution to identify an Fe storage site in Chlamydomonas, determine the spatial distribution of multiple essential trace elements in chlorophyte algae, and quantify acidocalcisomal metal content in single cells in situations of Fe and Cu overaccumulation. We took advantage of a Chlamydomonas vacuolar transporter chaperone (vtc1) mutant (defective in polyP synthesis and hence also Ca content) (43, 47) to distinguish the role of polyP and Ca in acidocalcisome Fe sequestration. XFM also enabled the comparison of single-cell trace metal quotas with bulk quantification of Cu and Fe in corresponding cell cultures, allowing us to distinguish the nutritional state for Cu and Fe in individual cells.