β-arrestin–dependent PI(4,5)P2 synthesis boosts GPCR endocytosis

β-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein–coupled receptors (GPCRs). Previous studies show that β-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P₂). We found that β-arrestin also helped promote synthesis of PI(4,5)P₂ and up-regulated GPCR endocytosis. We studied these questions with the G_q-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P₂, which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P_2, could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P₂ could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of β-arrestin revealed that endogenous β-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P₂-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of ∼90 s, 100 times longer than for unactivated receptors. PAR2/β-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, β-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P₂ generation that promotes recruitment of PI(4,5)P₂-dependent endocytic machinery.

Membrane phosphatidylinositide lipids (PPIs) are dynamic regulators of diverse cell functions, and their dysregulation underlies numerous human diseases (1). This paper concerns the key involvement of plasma membrane (PM) phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P₂) in refining receptor–G protein and receptor–β-arrestin coupling (2, 3) and preparing for the endocytosis of receptors (4). Endocytosis requires clustering of adapter proteins on the PM, nucleation of clathrin-coated membrane pits, capture of receptors with β-arrestin (5–7), and pinching off of pits as intracellular vesicles by dynamin GTPase (4, 8–10). In clathrin-mediated endocytosis, PI(4,5)P₂ is typically needed for the assembly of the adaptor protein complexes, clathrin-coated pits (CCPs), and dynamin complexes (4, 11–14). Hence, receptor internalization should be compromised if PI(4,5)P₂ pools are depleted. This raises the question of how receptors that signal by depleting PI(4,5)P₂ can still be internalized. In this study, we found roles of receptor stimulation and β-arrestin in promoting resynthesis of PI(4,5)P₂, thus enabling endocytosis at the PM.Synthesis of PPIs starts with phosphatidylinositol and families of lipid kinases that generate the mono-, bis-, and tris-phosphorylated inositol ring. PM phosphatidylinositol 4-phosphate (PI(4)P) and PI(4,5)P₂ are produced by several mechanisms potentially involving other membrane compartments. They can be synthesized by lipid 4-kinases acting on PM phosphatidylinositol and by lipid 5-kinases acting on PM PI(4)P; they can be delivered in exchange for other lipids by phosphatidylinositol exchange proteins; and they can be delivered through fusion with other membranes (15–23). Such studies show that the PPI pools in different membranes are interdependent (21). For example, depleting PI(4)P locally in the trans-Golgi using a recruitable PI(4)P 4-phosphatase tool reduces the generation of PI(4,5)P₂ at the PM (24). Conversely, depleting PI(4,5)P₂ at the PM by activating muscarinic or angiotensin II receptors also strongly decreases total cellular PI(4)P (25–27). New evidence is emerging that the PPI composition controls membrane trafficking between organelles. For instance, trafficking of mannose 6-phosphate receptors from the Golgi to the PM can be slowed by reduction of PPI synthesis (28) presumably because PPIs are important for fusion of receptor-containing vesicles with the PM.Here, we study contributions of PPI pools to the endocytosis of the G_q-coupled protease-activated receptor 2 (PAR2). This receptor is involved in inflammatory responses (29), sensation of inflammatory pain (30), and cancer metastasis (31). It has been a target of drug development (32) facilitated by recent crystal structures (33). Stimulation of this receptor activates phospholipase C (PLC) to cleave and deplete PI(4,5)P₂ with accompanying production of diacylglycerol, inositol trisphosphate, and calcium signals (34, 35). Activation of the PAR-receptor family has unique properties. The receptor is activated by cleavage of the N terminus by serine proteases such as thrombin, tryptase, or trypsin (34, 36), which generates a tethered N-terminal ligand. The activation stimulates G_q but is followed quickly by desensitization that terminates G_q signaling (34, 35, 37). Our previous experimental results and mathematical modeling suggest that rapid phosphorylation of PAR2 precedes desensitization and that β-arrrestin clamps the phosphorylated and ligand-bound state of the receptor, protecting it from dephosphorylation by serine/threonine phosphatases (38). Then, the receptor is internalized slowly via a clathrin- and dynamin-dependent pathway (8). This rapidly desensitizing receptor is well suited to address mechanisms involved in PPI lipid–dependent G_qPCR endocytosis.Using genetic and optical tools to manipulate and measure PI(4)P and PI(4,5)P₂ levels acutely at the Golgi or the PM, we now demonstrate that PAR2 internalization can be controlled by PM PI(4,5)P₂ that is replenished using both PM and Golgi pools of PI(4)P. A β-arrestin–dependent activation of PIP5-kinase (PIP5K) at the PM turned out to be critical in the formation of PI(4,5)P₂- and PI(4)P-requiring CCPs and potentially other endocytic machinery for receptor internalization.     

In situ organic Fenton-like catalysis triggered by anodic polymeric intermediates for electrochemical water purification

Organic Fenton-like catalysis has been recently developed for water purification, but redox-active compounds have to be ex situ added as oxidant activators, causing secondary pollution problem. Electrochemical oxidation is widely used for pollutant degradation, but suffers from severe electrode fouling caused by high-resistance polymeric intermediates. Herein, we develop an in situ organic Fenton-like catalysis by using the redox-active polymeric intermediates, e.g., benzoquinone, hydroquinone, and quinhydrone, generated in electrochemical pollutant oxidation as H₂O₂ activators. By taking phenol as a target pollutant, we demonstrate that the in situ organic Fenton-like catalysis not only improves pollutant degradation, but also refreshes working electrode with a better catalytic stability. Both ¹O₂ nonradical and ·OH radical are generated in the anodic phenol conversion in the in situ organic Fenton-like catalysis. Our findings might provide a new opportunity to develop a simple, efficient, and cost-effective strategy for electrochemical water purification.

The efficient generation of reactive oxygen species is essential for pollutant degradation in water purification. The metal-mediated Fenton catalysis has been widely used for several decades owning to its high efficiency, low cost, and easy operation (1). However, it has several technical drawbacks to largely limit further applications, e.g., harsh pH, metal-rich sludge, secondary pollution, and poor stability (1). Alternatively, the metal-free Fenton catalysis has recently attracted increasing interests. Redox-active compounds serve as the oxidant activator to decompose pollutants via radical and/or nonradical pathways (2–5). These pathways depend highly on the atomic and electronic structures and molecular configurations of compounds and their molecular interactions with oxidants (6–18). So far organic activators are ex situ introduced and cause secondary pollution, although the performance can be largely improved (2–18). Such an intrinsic drawback greatly restricts its practical applications. Thus, in situ organic Fenton-like catalysis without secondary pollution is greatly desired for clean and safe water purification.Electrochemical oxidation (EO) at low bias is widely used for pollutant degradation owning to its high current efficiency and low energy consumption, but largely suffers from electrode fouling (19, 20). Such fouling is mainly caused by anodic polymeric intermediates with large molecular size, low geometric polarity, and high structural stability, thus anodic oxidation is thermodynamically terminated at this stage (19, 20). How to remove polymeric intermediates is essential for electrochemical water purification. It is interesting to note that anodic polymeric intermediates usually contain quinonelike moieties (C = O) and persistent organic radicals, as the electrons in nucleophilic C-OH can be readily transferred to generate C-O· and C = O (19, 20). Quinonelike moieties are redox-active because of their high electron density and strong electron-donating properties, thus can serve as the metal ligand and reductant to enhance transition-metal redox cycling, and also be involved in the environmental geochemistry of natural organic matters (21–30). Moreover, quinonelike moieties and persistent organic radicals can directly serve as an organic activator to initiate organic Fenton-like catalysis for environmental remediation (31–40). Thus, these redox-active anodic polymeric intermediates are likely to trigger organic Fenton-like catalysis.Inspired by above analyses, we constructed and validated in situ organic Fenton-like catalysis for electrochemical water purification at low bias before oxygen evolution (Scheme 1). Phenol, a model chemical widely present in environments, and other typical halogenated and nonhalogenated aromatic compounds were selected as target pollutants. Carbon felt (CF), a model material with high activity and low cost, and other typical dimensionally stable anodes were selected as target electrodes. Reaction systems were named in the form of “EO + ex situ added reagent + cathode,” as their anodes were identical. Pollutant degradation and electrode antifouling performances were evaluated under various conditions. After the major reactive oxygen species were identified using a suite of testing methods, and the potential role of trace transition metals, especially iron and copper, was examined, the possible molecular mechanism of the in situ organic Fenton-like catalysis was proposed.Open in a separate windowScheme 1.Scheme diagrams of the EO-Ti, EO/H₂O₂-Ti, and EO/O₂-CF systems.     

PIP2 corrects cerebral blood flow deficits in small vessel disease by rescuing capillary Kir2.1 activity

Cerebral small vessel diseases (SVDs) are a central link between stroke and dementia—two comorbidities without specific treatments. Despite the emerging consensus that SVDs are initiated in the endothelium, the early mechanisms remain largely unknown. Deficits in on-demand delivery of blood to active brain regions (functional hyperemia) are early manifestations of the underlying pathogenesis. The capillary endothelial cell strong inward-rectifier K⁺ channel Kir2.1, which senses neuronal activity and initiates a propagating electrical signal that dilates upstream arterioles, is a cornerstone of functional hyperemia. Here, using a genetic SVD mouse model, we show that impaired functional hyperemia is caused by diminished Kir2.1 channel activity. We link Kir2.1 deactivation to depletion of phosphatidylinositol 4,5-bisphosphate (PIP₂), a membrane phospholipid essential for Kir2.1 activity. Systemic injection of soluble PIP₂ rapidly restored functional hyperemia in SVD mice, suggesting a possible strategy for rescuing functional hyperemia in brain disorders in which blood flow is disturbed.

Cerebral small vessel diseases (SVDs), a seemingly intractable ensemble of genetic and sporadic diseases that progress silently for years before becoming clinically symptomatic, have emerged as a central link between stroke and dementia—two comorbidities that rank among the most pressing human health issues (1, 2). SVDs are responsible for more than 25% of ischemic strokes (1) and are the leading cause of age-related cognitive decline and disability, accounting for more than 40% of dementia cases (1, 3). Despite the enormous impact of SVDs on human health, the disease processes and key biological mechanisms underlying these disorders remain largely unknown. Notably, there are no specific treatments for sporadic or genetic SVDs (4).In the healthy brain, cerebral blood flow (CBF) is controlled so as to meet the changing demands of active neurons. This activity-dependent blood-delivery process (functional hyperemia) is rapidly and precisely controlled through a number of molecular mechanisms collectively termed “neurovascular coupling.” We demonstrated that brain capillaries act as a neural activity-sensing network, showing that brain capillary endothelial cells (cECs) are capable of initiating an electrical (hyperpolarizing) signal in response to neural activity that propagates retrogradely to dilate upstream feeding arterioles, thereby increasing local blood flow. We have further established that extracellular K⁺—a byproduct of every neuronal action potential—is the critical mediator of this process and have identified the cEC strong inward-rectifier K⁺ channel, Kir2.1, as the target of K⁺ ions (5–7). Notably, we have further shown that phosphatidylinositol 4,5-bisphosphate (PIP₂), a minor inner leaflet phospholipid, is an important physiological regulator of cEC Kir2.1 channels (8–10), a finding in accord with previous studies demonstrating the requirement of PIP₂ for channel activity (11, 12).Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL ), caused by highly stereotyped mutations in the extracellular domain of the NOTCH3 receptor, is the most common monogenic inherited form of SVD and a model for more frequent sporadic forms (2, 3). We recently demonstrated that defective functional hyperemia is an early disturbance in the TgNotch3^R169C mouse model of CADASIL (13), which overexpresses a mutation identified in humans (14). Notably, CADASIL patients exhibit deficits in functional hyperemia long before evidence of significant disability or cognitive decline (15). Our recent studies using the TgNotch3^R169C mouse model (hereafter, SVD mice) implicate altered extracellular matrix dynamics in this disease. Specifically, these studies showed that the matrix metalloproteinase inhibitor TIMP3, which accumulates in NOTCH3 extracellular domain (NOTCH3^ECD) deposits surrounding vascular smooth muscle cells (SMCs) and pericytes, suppresses the activity of the matrix metalloproteinase ADAM17, inhibiting shedding of the epidermal growth factor receptor (EGFR) ligand heparin-binding EGF-like growth factor (HB-EGF), thereby decreasing EGFR activity (13, 16–19). One direct consequence of this decreased ADAM17/HB-EGF/EGFR signaling is a reduction in functional hyperemia (13). However, the basis of this deficit is not known. Here, we found that a mechanism—Kir2.1 channel down-regulation in cECs—operates to abrogate capillary-to-arteriole electrical signaling and cause functional hyperemia deficits.     

Goblet cell LRRC26 regulates BK channel activation and protects against colitis in mice

Goblet cells (GCs) are specialized cells of the intestinal epithelium contributing critically to mucosal homeostasis. One of the functions of GCs is to produce and secrete MUC2, the mucin that forms the scaffold of the intestinal mucus layer coating the epithelium and separates the luminal pathogens and commensal microbiota from the host tissues. Although a variety of ion channels and transporters are thought to impact on MUC2 secretion, the specific cellular mechanisms that regulate GC function remain incompletely understood. Previously, we demonstrated that leucine-rich repeat-containing protein 26 (LRRC26), a known regulatory subunit of the Ca²⁺-and voltage-activated K⁺ channel (BK channel), localizes specifically to secretory cells within the intestinal tract. Here, utilizing a mouse model in which MUC2 is fluorescently tagged, thereby allowing visualization of single GCs in intact colonic crypts, we show that murine colonic GCs have functional LRRC26-associated BK channels. In the absence of LRRC26, BK channels are present in GCs, but are not activated at physiological conditions. In contrast, all tested MUC2⁻ cells completely lacked BK channels. Moreover, LRRC26-associated BK channels underlie the BK channel contribution to the resting transepithelial current across mouse distal colonic mucosa. Genetic ablation of either LRRC26 or BK pore-forming α-subunit in mice results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate. These results demonstrate that normal potassium flux through LRRC26-associated BK channels in GCs has protective effects against colitis in mice.

The colonic epithelium is composed of a single layer of heterogeneous cells, covered by mucus, that separate the luminal contents from host tissues. Acting both in concert and individually, the diverse cells comprising the epithelial layer play the functions of protection (1), sensation (2, 3), transport of substances (4, 5), and repair (6). Colonic epithelial cells belong to three lineages: Absorptive enterocytes, enteroendocrine cells, and goblet cells (GCs). The colonic epithelium is morphologically organized into repeating units called crypts of Lieberkühn, where stem cells located at the base of the crypts divide and successively differentiate into the mature lineages as they migrate toward the crypt surface (7). Many of the key specialized functions of epithelial cells are, in part, defined by proteins involved in ion transport, located either on their luminal or basolateral membrane. Thus, among different gastrointestinal epithelial cells, ion channels, carriers, exchangers, and pumps work in concert to define a variety of essential functions: 1) Solute and electrolyte absorption and secretion in absorptive enterocytes (reviewed in refs. 5 and 8); 2) environment sensation and serotonin secretion by enteroendocrine cells (2, 9); and 3) mucus secretion by GCs and subsequent mucus maturation into the protective layer covering the epithelial surface (10–12). Despite this progress, ionic transport in GCs and its implications in GC physiology is a topic that remains poorly understood. Here, we address the role of the Ca²⁺- and voltage-activated K⁺ channel (BK channel) in GCs.GCs play two primary roles: One related to the maintenance of the mucosal barrier (reviewed in refs. 1 and 13) and one related with the mucosal immune homeostasis (reviewed in refs. 14 and 15). The role of GCs in barrier maintenance consists in generation of the mucus layer lining the intestinal lumen. One way GCs carry out this role is by secreting MUC2, the gel-forming mucin that forms the scaffold of the mucus layer separating luminal pathogens and commensal microbiota from the epithelial surface (11, 12, 15, 16). This separation is critical, as has been demonstrated in both animal models and humans: Mouse models with deficient mucus layer generation develop spontaneous colitis (16, 17), whereas a more penetrable mucus layer has been observed in patients with ulcerative colitis (UC), a form of human inflammatory bowel disease (IBD) (18, 19). The constant replenishment of the mucus layer involves MUC2 exocytosis from GCs, and subsequent maturation (hydration and expansion) of the secreted MUC2 to form the gel-like mucus coating the epithelium (15). Both exocytosis and maturation of MUC2 are highly dependent on anion and K⁺ transport (10–12, 20). It has been proposed that mucin exocytosis in colon requires activities of the Na⁺/K⁺/2Cl⁻ cotransporter (NKCC1) (20, 21), and also anion and K⁺ channels whose identities are still unclear (20). It is also not clearly known whether specific ionic conductances are intrinsic to GCs or are located in the surrounding absorptive enterocytes. Although several types of K⁺ channels—including K_Ca3.1, Kv7.1, and BK channels—have been found in colonic epithelial cells (22–27), to what extent any of those K⁺ channels are specifically associated with GCs or critical to their function remains unclear. To date, most functional studies about colonic K⁺ channels have focused on their roles in electrolyte and fluid secretion/absorption of the whole colon, whereas the cellular events relating K⁺ channels to specific roles in GC function are still poorly understood.Among colonic epithelial K⁺ channels, the BK channel (also known as K_Ca1.1), the Ca²⁺- and voltage-activated K⁺ channel of high conductance, has been proposed to be the main component of colonic K⁺ secretion into the lumen (28–30). BK channels are homotetramers of the pore-forming BKα subunit, but can also contain tissue-specific regulatory subunits that critically define the functional properties of the channel (31). BK channels composed exclusively of the pore-forming BKα subunit are unlikely to be activated at the physiological conditions of epithelial cells and, as a consequence, the molecular properties of colonic BK channels that would allow them to contribute to colonic ion transport remain unclear. Recently, we established that the leucine-rich repeat-containing protein 26 (LRRC26), a BK regulatory γ-subunit, is specifically expressed in secretory epithelial cells, including GCs of the gastrointestinal tract (32). When LRRC26 is present in a BK channel complex, the resulting channel activates near normal resting physiological conditions, even in the absence of any elevation of intracellular Ca²⁺ (33).In the present study, we have specifically probed the role of BK channels in cells of the colonic epithelium and examined the impact of deletions of either the BKα subunit or LRRC26 on colonic function. Here, through recordings from identified GCs in intact colonic crypts, we show that LRRC26-associated BK channels contribute the major K⁺ current at low intracellular Ca²⁺ (∼250 nM) in mouse colonic GCs. Furthermore, the LRRC26-containing BK channels are activated near −40 mV, even in the absence of intracellular Ca²⁺. In contrast, in identified GCs from Lrrc26^−/− mice, BK current is present, but it is only activated at membrane potentials unlikely to ever occur physiologically. Surprisingly, all colonic epithelial MUC2⁻ cells sampled completely lack functional BK channels. To establish that the LRRC26-containing BK channels contribute to normal K⁺ fluxes in intact colon tissue, we show that the transepithelial current across distal colon at rest has a component dependent on LRRC26-associated BK channels, which is absent when either BKα or LRRC26 is genetically deleted. Moreover, the genetic ablation of either LRRC26 or BK channel results in a dramatically enhanced susceptibility to colitis induced by dextran sodium sulfate (DSS). Overall, our results suggest that normal potassium flux through LRRC26-associated BK channels in GCs has a protective role against development of colitis.     

Stimulation of soil respiration by elevated CO2 is enhanced under nitrogen limitation in a decade-long grassland study

Whether and how CO₂ and nitrogen (N) availability interact to influence carbon (C) cycling processes such as soil respiration remains a question of considerable uncertainty in projecting future C–climate feedbacks, which are strongly influenced by multiple global change drivers, including elevated atmospheric CO₂ concentrations (eCO₂) and increased N deposition. However, because decades of research on the responses of ecosystems to eCO₂ and N enrichment have been done largely independently, their interactive effects on soil respiratory CO₂ efflux remain unresolved. Here, we show that in a multifactor free-air CO₂ enrichment experiment, BioCON (Biodiversity, CO₂, and N deposition) in Minnesota, the positive response of soil respiration to eCO₂ gradually strengthened at ambient (low) N supply but not enriched (high) N supply for the 12-y experimental period from 1998 to 2009. In contrast to earlier years, eCO₂ stimulated soil respiration twice as much at low than at high N supply from 2006 to 2009. In parallel, microbial C degradation genes were significantly boosted by eCO₂ at low but not high N supply. Incorporating those functional genes into a coupled C–N ecosystem model reduced model parameter uncertainty and improved the projections of the effects of different CO₂ and N levels on soil respiration. If our observed results generalize to other ecosystems, they imply widely positive effects of eCO₂ on soil respiration even in infertile systems.

Elevation of atmospheric CO₂ concentrations, owing to fossil fuel combustion and land-use changes, represents one of the greatest scientific and political concerns of the 21st century (1). Carbon (C) movement into the atmosphere annually from soils (i.e., soil CO₂ efflux or soil respiration) is much larger than annual C emissions from fossil fuel combustion (2), and thus even small changes in soil respiration could have significant impacts on the pace of change in atmospheric CO₂. Numerous studies have demonstrated that elevated CO₂ (eCO₂) has a direct stimulatory effect on rates of plant photosynthesis (3), and an indirect positive effect on soil respiration, which typically includes autotrophic respiration from plant roots and heterotrophic respiration from microbial decomposition of litter and soil organic matter (SOM). The eCO₂ stimulatory effect on soil respiration is commonly attributed to the following three mutually nonexclusive mechanisms from the actions of plants and microorganisms (4–7): enhanced root respiration associated with greater belowground plant biomass, enhanced microbial decomposition of fresh C due to greater supply of foliar and root-derived labile soil C, and increased microbial priming of old SOM fueled by this increased supply of labile soil C (4, 5). The stimulation of soil respiration by eCO₂ (7, 8) has the potential to greatly accelerate the future rate of increase in atmospheric CO₂ concentrations unless matched by an offsetting increase in net C uptake.Human activities have also increased nitrogen (N) deposition to natural ecosystems (9). N enrichment is a growing concern because it disturbs N-cycle processes in many ecosystems (9). Various studies have suggested that N addition can either increase (10, 11) or reduce (12–15) soil CO₂ efflux, while other studies have suggested that N addition does not influence soil CO₂ efflux (16, 17), depending on ecosystem type and season of the year.The stimulation of soil respiration by eCO₂ also could be strongly influenced by variability in ambient soil N availability and the rate of atmospheric N deposition (18). However, studies that have explored the interactive effects of eCO₂ and N on soil respiration are extremely scarce. For instance, an open-top study of young subtropical tree seedlings in contrasting eCO₂ and N treatments in transplanted soil found that response to eCO₂ was enhanced by high levels of N addition (10 g⋅m⁻²⋅y⁻¹) in the earliest 2 y but unaffected by the same N supply in the subsequent year (19, 20). A free-air enrichment study in perennial grasslands also found no interaction between eCO₂ and N addition treatments over the first 2 y of the study (21). Given that many questions about such potential interactions remain unresolved (22), here we report on 12 y of results in that same grassland study, assessing whether interactions develop and, if so, what underlying mechanisms might drive them.It is well known that N availability alters many aspects of ecosystems (12, 23, 24) and thus could hypothetically influence responses of soil respiration to eCO₂. Three potentially off-setting and interrelated mechanisms have been proposed. First, N limitation could affect belowground productivity and thus root respiration. For example, if N limitation constrains plant canopy development and the stimulatory effect of eCO₂ on photosynthesis, and thus limits total productivity belowground, root respiration will decline (24). On the other hand, the same N limitation constraint on canopy development combined with stimulatory effects of eCO₂ on photosynthesis could increase plant investment of C in nutrient-absorbing systems (25, 26), favoring C allocation to roots at the expense of aboveground biomass. Such a shift in allocation could increase root respiration (27). Second, changes in root detrital production and exudation of labile C into soils can influence substrate supply that fuels soil microbial activity and heterotrophic respiration. Third, the supply of labile C into soils can influence decomposition of SOM through the priming effect, which would also influence soil heterotrophic respiration (28). Under N limitation, greater photosynthesis caused by eCO₂ could stimulate mining of N from SOM, and thus soil heterotrophic respiration, through enhanced priming mechanisms (29).Although various studies indicate that N availability plays critical roles in mediating soil respiration (10–17, 23, 30, 31), divergent results are observed: positive (10, 11, 23), neutral (16, 17, 30), or negative (12–15, 30, 31). Thus, the impacts of N availability on the magnitude and duration of the eCO₂ enhancement of soil respiration and its underlying mechanisms remain elusive, particularly under field settings. In addition, recent modeling efforts demonstrated the importance of understanding microbial C decomposition for more confidently extrapolating soil C cycling processes (32, 33). However, to date, it remains uncertain whether and how microbial processes influence the responses of terrestrial ecosystems to eCO₂ and N deposition and how best to incorporate information regarding microbial responses to eCO₂ and N into climate-C models for better simulation and prediction (32, 34, 35).Herein, we report results from a well-replicated long-term (12 y at the time of sampling) CO₂ × N experiment, BioCON (Biodiversity, CO₂, and N deposition) (24), to elucidate the interactive effects of eCO₂ and N enrichment on soil respiration and their underlying mechanisms. From 1998 to 2009, we measured soil CO₂ efflux and other biogeochemical processes on 296 plots containing different numbers (1, 4, 9, or 16 species) and combinations (C₃ and C₄ grasses, forbs, and legumes) of perennial plant species at ambient CO₂ (aCO₂) or eCO₂ (+180 ppm) with either ambient N supply (aN) or enriched N supply (eN, i.e., +4 g N⋅m⁻²⋅y⁻¹). Hereafter, we refer to these four treatment combinations as aCO₂-aN, eCO₂-aN, aCO₂-eN, and eCO₂-eN. The contrasting high versus low levels of N supply in this study was a rough proxy for a part of the worldwide range of N supply rates in soils as well as for times or places with low versus high N deposition (24). Thus, we posit that the results are relevant to understanding the potentially different responses to eCO₂ of both low versus high N fertility soils and contexts with low versus high N deposition. In 2009, we also assessed responses of microbial community functional gene structure to eCO₂ and N enrichment to gain insights into microbial regulation of soil respiration. In addition, we incorporated microbial functional trait information into ecosystem models to explore means of better prediction of C cycling. Our overarching hypothesis is that N limitation would accelerate the stimulatory effects of eCO₂ on soil respiration, primarily via microbial N mining mechanisms. We further explored the possibility that microbial functional trait information would greatly help to constrain the uncertainty of model parameters and hence significantly improve confidence in model simulations and predictions.     

Pharmacological inhibition of PI5P4Kα/β disrupts cell energy metabolism and selectively kills p53-null tumor cells

Most human cancer cells harbor loss-of-function mutations in the p53 tumor suppressor gene. Genetic experiments have shown that phosphatidylinositol 5-phosphate 4-kinase α and β (PI5P4Kα and PI5P4Kβ) are essential for the development of late-onset tumors in mice with germline p53 deletion, but the mechanism underlying this acquired dependence remains unclear. PI5P4K has been previously implicated in metabolic regulation. Here, we show that inhibition of PI5P4Kα/β kinase activity by a potent and selective small-molecule probe disrupts cell energy homeostasis, causing AMPK activation and mTORC1 inhibition in a variety of cell types. Feedback through the S6K/insulin receptor substrate (IRS) loop contributes to insulin hypersensitivity and enhanced PI3K signaling in terminally differentiated myotubes. Most significantly, the energy stress induced by PI5P4Kαβ inhibition is selectively toxic toward p53-null tumor cells. The chemical probe, and the structural basis for its exquisite specificity, provide a promising platform for further development, which may lead to a novel class of diabetes and cancer drugs.

There are two synthetic routes for phosphatidylinositol 4,5-bisphosphate, or PI(4,5)P₂, a versatile phospholipid with both structural and signaling functions in most eukaryotic cells (1 –3). The bulk of PI(4,5)P₂ is found at the inner leaflet of the plasma membrane and is synthesized from phosphatidylinositol 4-phosphate, or PI(4)P, by type 1 phosphatidylinositol phosphate kinase PI4P5K (4, 5). A smaller fraction of PI(4,5)P₂ is generated from the much rarer phosphatidylinositol 5-phosphate, or PI(5)P, through the activity of type 2 phosphatidylinositol phosphate kinase PI5P4K (6, 7). Although PI5P4K is as abundantly expressed as PI4P5K (8), its function is less well understood (9). It has been proposed that PI5P4K may play a role in suppressing PI(5)P, which is often elevated by stress (10, 11), or produce local pools of PI(4,5)P₂ at subcellular compartments such as Golgi and nucleus (12).Higher animals have three PI5P4K isoforms, α, β, and γ, which are encoded by three different genes, PIP4K2A, PIP4K2B, and PIP4K2C. The three isoforms differ, at least in vitro, significantly in enzymatic activity: PI5P4Kα is two orders of magnitude more active than PI5P4Kβ, while PI5P4K-γ has very little activity (13). PI5P4Ks are dimeric proteins (14), and the possibility that they can form heterodimers may have important functional implications, especially for the lesser active isoforms (15, 16). PI5P4Kβ is the only isoform that preferentially localizes to the nucleus (17).Genetic studies have implicated PI5P4Kβ in metabolic regulation (18, 19). Mice with both PIP4K2B genes inactivated manifest hypersensitivity to insulin stimulation (adult males are also leaner). Although this is consistent with the observation that PI(5)P levels, which can be manipulated by overexpressing PI5P4K or a bacterial phosphatase that robustly produces PI(5)P from PI(4,5)P₂, correlate positively with PI3K/Akt signaling, the underlying molecular mechanisms remain undefined (20). Both male and female PIP4K2B ^−/− mice are mildly growth retarded. Inactivation of the only PI5P4K isoform in Drosophila also produced small and developmentally delayed animals (21). These phenotypes may be related to suppressed TOR signaling (22, 23), but again, the underlying mechanism is unclear since TORC1 is downstream of, and positively regulated by, PI3K/Akt. Knocking out the enzymatically more active PI5P4Kα, in contrast, did not produce any overt metabolic or developmental phenotypes (19).Malignant transformation is associated with profound changes in cell metabolism (24, 25). Although metabolic reprograming generally benefits tumor cells by increasing energy and material supplies, it can also, counterintuitively, generate unique dependencies (26, 27). Loss of p53, a tumor suppressor that is mutated in most human cancers, has been shown to render cells more susceptible to nutrient stress (28, 29) and to the antidiabetic drug metformin (30, 31). Although TP53 ^−/− and PIP4K2B ^−/− mice are themselves viable, combining the two is embryonically lethal (19). Knocking out three copies of PI5P4K (PIP4K2A ^−/− PIP4K2B ^+/− ) greatly reduces tumor formation and cancer-related death in TP53 ^−/− animals (19). The synthetic lethal interaction between p53 and PI5P4Kα/β was thought to result from suppressed glycolysis and increased reactive oxygen species (19), although how the lipid kinases impact glucose metabolism remains uncertain.Given the interest in the physiological function of this alternative synthetic route for PI(4,5)P₂, and the potential of PI5P4K inactivation in treating type 2 diabetes and cancer, several attempts have been made to identify chemical probes that target various PI5P4K isoforms, which yielded compounds with micromolar affinity and unknown selectivity (32 –35). Here, we report the development of a class of PI5P4Kα/β inhibitors that have much improved potency and better-defined selectivity. Using the chemical probe, we show that transient inhibition of the lipid kinases alters cell energy metabolism and induces different responses in muscle and cancer cells.     

Direct and continuous generation of pure acetic acid solutions via electrocatalytic carbon monoxide reduction

Electrochemical CO₂ or CO reduction to high-value C₂₊ liquid fuels is desirable, but its practical application is challenged by impurities from cogenerated liquid products and solutes in liquid electrolytes, which necessitates cost- and energy-intensive downstream separation processes. By coupling rational designs in a Cu catalyst and porous solid electrolyte (PSE) reactor, here we demonstrate a direct and continuous generation of pure acetic acid solutions via electrochemical CO reduction. With optimized edge-to-surface ratio, the Cu nanocube catalyst presents an unprecedented acetate performance in neutral pH with other liquid products greatly suppressed, delivering a maximal acetate Faradaic efficiency of 43%, partial current of 200 mA⋅cm⁻², ultrahigh relative purity of up to 98 wt%, and excellent stability of over 150 h continuous operation. Density functional theory simulations reveal the role of stepped sites along the cube edge in promoting the acetate pathway. Additionally, a PSE layer, other than a conventional liquid electrolyte, was designed to separate cathode and anode for efficient ion conductions, while not introducing any impurity ions into generated liquid fuels. Pure acetic acid solutions, with concentrations up to 2 wt% (0.33 M), can be continuously produced by employing the acetate-selective Cu catalyst in our PSE reactor.

Electrochemically reducing carbon dioxide (CO₂) or carbon monoxide (CO) to commodity fuels or chemicals, with the input of green and economical renewable electricity, has become an alternative route to traditional chemical engineering processes (1–7). To date, a variety of catalysts have been demonstrated to reduce CO₂ into C₁ products (CO, formate, etc.) with high Faradaic efficiencies (FEs) (6, 8–14); however, the critical step of C-C coupling toward high-value C₂₊ products was rarely observed on catalysts other than Cu (15–20). Due to its proper binding strength with *CO (19, 21, 22), Cu has shown the capability of catalyzing the coupling of two carbons or more in CO₂/CO reduction reactions (CO₂RR/CORR), which, however, typically suffers from large overpotential and low selectivity (23–25). In previous studies on Cu catalysts, tremendous efforts have been focused on how to promote C-C coupling toward high selectivity of C₂₊ products, including facet engineering (26–28), grain boundary engineering (29, 30), surface modifications (31–33), etc. For example, recent studies on facet dependence of C-C coupling have shown that the C₂₊ selectivity could be improved on Cu(100) compared to other facets due to more stabilized *OCCO intermediates (26, 34). While the overall C₂₊ selectivity is constantly improved by researchers in this field, the generated products are typically a mixture of different C₂₊ compounds, which subsequently necessitates energy- and cost-intensive downstream product separation and purification processes (35–37). A solution to continuously obtain a highly pure, single C₂₊ product stream, which can be directly fed into practical applications, will impose a significant scientific and economic impact but has been rarely studied before.Ethylene, ethanol, and acetate are the three major C₂ products that have been widely reported in CO₂RR/CORR on Cu materials (16–18, 38–44). Compared to gas-phase products, liquid products show significant advantages due to their high energy densities and ease of storage and distribution (45). Nevertheless, the generation of a high-purity C₂ liquid fuel via electrochemical CO₂RR/CORR is challenging due to the involvement of two types of impurities. First, in previous studies of Cu catalysts, while the C₂ product can be dramatically increased by strategies such as exposing (100) facets, ethanol and acetic acid usually share the same potential window and are cogenerated simultaneously, making it difficult to obtain a single C₂ liquid product (29, 46, 47); Second, in traditional H-cell or flow-cell reactors, the generated liquid products were mixed with solutes in liquid electrolytes such as KOH or KHCO₃, which requires extra separation and concentration processes to recover pure liquid fuel solutions in practical applications (37, 48, 49). An integration of rational design in both catalytic material and reactor, for high single-C₂ selectivity and electrolyte-free liquid fuel output, respectively, therefore becomes the key to achieve the goal of pure C₂ liquid fuels.Here we report the continuous generation of pure acetic acid solutions via CORR on Cu nanocube (NC) catalysts in a porous solid electrolyte (PSE) reactor. By flexibly tuning the edge to (100) surface ratio, the medium-size Cu NC catalyst exhibits a maximal acetate Faradaic efficiency of 43% with a partial current density of ∼200 mA⋅cm⁻² in neutral pH, setting up a different acetate performance benchmark (18, 40, 41). More importantly, the selectivity of other liquid products (ethanol and trace amount of n-propanol) was suppressed to below 2%, suggesting an ultrahigh acetic acid relative purity of up to 98 wt%. An impressive stability was demonstrated by a continuous operation of CORR under 150 mA⋅cm⁻² current for over 150 h with negligible degradation in selectivity and activity. This acetate-selective Cu NC catalyst was successfully employed into our PSE reactor for the continuous generation of electrolyte-free, pure acetic acid solutions, with overall current of up to 1 A⋅cm⁻² and acetic acid relative purity as high as 96 wt%. Different from the traditional liquid electrolyte, our PSE layer can efficiently conduct ions while it does not introduce any impurity ions into the generated liquid products. Density functional theory (DFT) calculations suggest that the stepped edge sites on the Cu NC readily dissociate *OCCOH into *CCO and *OH, which is a key step in promoting the formation of acetate. We propose that the large NC has a low edge-to-facet ratio and therefore cogenerates considerable ethanol and ethylene instead of acetate, while a small NC provides less active (100) area for C-C coupling and therefore predominantly catalyzes the hydrogen evolution reaction (HER) instead of yielding C₂ products.     

High-dimensional mass cytometry analysis of NK cell alterations in AML identifies a subgroup with adverse clinical outcome

Natural killer (NK) cells are major antileukemic immune effectors. Leukemic blasts have a negative impact on NK cell function and promote the emergence of phenotypically and functionally impaired NK cells. In the current work, we highlight an accumulation of CD56⁻CD16⁺ unconventional NK cells in acute myeloid leukemia (AML), an aberrant subset initially described as being elevated in patients chronically infected with HIV-1. Deep phenotyping of NK cells was performed using peripheral blood from patients with newly diagnosed AML (n = 48, HEMATOBIO cohort, {"type":"clinical-trial","attrs":{"text":"NCT02320656","term_id":"NCT02320656"}}NCT02320656) and healthy subjects (n = 18) by mass cytometry. We showed evidence of a moderate to drastic accumulation of CD56⁻CD16⁺ unconventional NK cells in 27% of patients. These NK cells displayed decreased expression of NKG2A as well as the triggering receptors NKp30 and NKp46, in line with previous observations in HIV-infected patients. High-dimensional characterization of these NK cells highlighted a decreased expression of three additional major triggering receptors required for NK cell activation, NKG2D, DNAM-1, and CD96. A high proportion of CD56⁻CD16⁺ NK cells at diagnosis was associated with an adverse clinical outcome and decreased overall survival (HR = 0.13; P = 0.0002) and event-free survival (HR = 0.33; P = 0.018) and retained statistical significance in multivariate analysis. Pseudotime analysis of the NK cell compartment highlighted a disruption of the maturation process, with a bifurcation from conventional NK cells toward CD56⁻CD16⁺ NK cells. Overall, our data suggest that the accumulation of CD56⁻CD16⁺ NK cells may be the consequence of immune escape from innate immunity during AML progression.

Natural killer (NK) cells are critical cytotoxic effectors involved in leukemic blast recognition, tumor cell clearance, and maintenance of long-term remission (1). NK cells directly kill target cells without prior sensitization, enabling lysis of cells stressed by viral infections or tumor transformation. NK cells are divided into different functional subsets according to CD56 and CD16 expression (2–4). CD56^bright NK cells are the most immature NK cells found in peripheral blood. This subset is less cytotoxic than mature NK cells and secretes high amounts of chemokines and cytokines such as IFNγ and TNFα. These cytokines have a major effect on the infected or tumor target cells and play a critical role in orchestration of the adaptive immune response through dendritic cell activation. CD56^dimCD16⁺ NK cells, which account for the majority of circulating human NK cells, are the most cytotoxic NK cells. NK cell activation is finely tuned by integration of signals from inhibitory and triggering receptors, in particular, those of NKp30, NKp46 and NKp44, DNAM-1, and NKG2D (5). Upon target recognition, CD56^dimCD16⁺ NK cells release perforin and granzyme granules and mediate antibody-dependent cellular cytotoxicity through CD16 (FcɣRIII) to clear transformed cells.NK cells are a major component of the antileukemic immune response, and NK cell alterations have been associated with adverse clinical outcomes in acute myeloid leukemia (AML) (6–9). Therefore, it is crucial to better characterize AML-induced NK cell alterations in order to optimize NK cell–targeted therapies. During AML progression, NK cell functions are deeply altered, with decreased expression of NK cell–triggering receptors and reduced cytotoxic functions as well as impaired NK cell maturation (6, 9–13). Cancer-induced NK cell impairment occurs through various mechanisms of immune escape, including shedding and release of ligands for NK cell–triggering receptors; release of immunosuppressive soluble factors such as TGFβ, adenosine, PGE2, or L-kynurenine; and interference with NK cell development, among others (14).Interestingly, these mechanisms of immune evasion are also seen to some extent in chronic viral infections, notably HIV (2). In patients with HIV, NK cell functional anergy is mediated by the release of inflammatory cytokines and TGFβ, the presence of MHC^low target cells, and the shedding of ligands for NK cell–triggering receptors (2). As a consequence, some phenotypical alterations described in cancer patients are also induced by chronic HIV infections, with decreased expression of major triggering receptors such as NKp30, NKp46, and NKp44 (15, 16); decreased expression of CD16 (17); and increased expression of inhibitory receptors such as T cell immunoreceptor with Ig and ITIM domains (TIGIT) (18) all observed. In addition, patients with HIV display an accumulation of CD56⁻CD16⁺ unconventional NK cells, a highly dysfunctional NK cell subset (19, 20). Mechanisms leading to the loss of CD56 are still poorly described, and the origin of this subset of CD56⁻ NK cells is still unknown. To date, two hypotheses have been considered: CD56⁻ NK cells could be terminally differentiated cells arising from a mixed population of mature NK cells with altered characteristics or could expand from a pool of immature precursor NK cells (21). Expansion of CD56⁻CD16⁺ NK cells is mainly observed in viral noncontrollers (19, 20). Indeed, CD56 is an important adhesion molecule involved in NK cell development, motility, and pathogen recognition (22–27). CD56 is also required for the formation of the immunological synapse between NK cells and target cells, lytic functions, and cytokine production (26, 28). As a consequence, CD56⁻CD16⁺ NK cells display lower degranulation capacities and decreased expression of triggering receptors, perforin, and granzyme B, dramatically reducing their cytotoxic potential, notably against tumor target cells (2, 19, 20, 29, 30). In line with this loss of the cytotoxic functions against tumor cells, patients with concomitant Burkitt lymphoma and Epstein-Barr virus infection display a dramatic increase of CD56⁻CD16⁺ NK cells (30), which could represent an important hallmark of escape to NK cell immunosurveillance in virus-driven hematological malignancies.To our knowledge, this population has not been characterized in the context of nonvirally induced hematological malignancies. In the present work, we investigated the presence of this population of unconventional NK cells in patients with AML, its phenotypical characteristics, and the consequences of its accumulation on disease control. Finally, we explored NK cell developmental trajectories leading to the emergence of this phenotype.     

PI(3,4)P2-mediated membrane tubulation promotes integrin trafficking and invasive cell migration

Invadopodia are integrin-mediated adhesions with abundant PI(3,4)P₂. However, the functional role of PI(3,4)P₂ in adhesion signaling remains unclear. Here, we find that the PI(3,4)P₂ biogenesis regulates the integrin endocytosis at invadopodia. PI(3,4)P₂ is locally produced by PIK3CA and SHIP2 and is concentrated at the trailing edge of the invadopodium arc. The PI(3,4)P₂-rich compartment locally forms small puncta (membrane buds) in a SNX9-dependent manner, recruits dynein activator Hook1 through AKTIP, and rearranges into micrometer-long tubular invaginations (membrane tubes). The uncurving membrane tube extends rapidly, follows the retrograde movement of dynein along microtubule tracks, and disconnects from the plasma membrane. Activated integrin-beta3 is locally internalized through the pathway of PI(3,4)P₂-mediated membrane invagination and is then actively recycled. Blockages of PI3K, SHIP2, and SNX9 suppress integrin-beta3 endocytosis, delay adhesion turnover, and impede transwell invasion of MEF-Src and MDA-MB-231 cells. Thus, the production of PI(3,4)P₂ promotes invasive cell migration by stimulating the trafficking of integrin receptor at the invadopodium.

Upon binding to the extracellular matrix, the integrin receptor acts as the transmembrane anchor of cell–matrix adhesion and recruits signal regulation factors to coordinate cytoskeletal reorganization and subsequent cell motility (1, 2). Invadopodia are specialized integrin-mediated adhesions that are utilized by invasive cancer cells to migrate through tissue barriers (3, 4). The invadopodium contains a core of densely polymerized F-actin that is surrounded by integrin receptors. Matrix metalloproteinases also accumulate at the invadopodium, degrade the extracellular matrix in proximity, and support cell invasion. The dynamic distribution of integrin receptors plays important roles in adhesion formation and cell migration (5, 6). In particular, the turnover of preexisting adhesions and the assembly of new ones have critical impacts on the speed and the persistence of cell migration. Microtubule targeting to the adhesion site and the associated motor activity can stimulate the bidirectional trafficking of cell surface receptors and modulate the adhesion composition (7, 8). The endocytosis of integrin receptors can weaken the preexisting adhesion and promote the adhesion disassembly. On the other hand, the redistribution of endocytosed integrin receptors back to the plasma membrane allows the formation of new adhesion sites and further supports the cell migration (9, 10).Phosphoinositide lipids are important signaling molecules in the plasma membrane and cytoplasmic organelles (11). The phosphorylation at the 3, 4, and/or 5 hydroxyl positions of the inositol head group can result in seven types of phosphoinositide lipids, including phosphatidylinositol monophosphate [PI(3)P, PI(4)P, and PI(5)P], diphosphate [PI(3,4)P₂, PI(3,5)P₂, and PI(4,5)P₂], and triphosphate [PI(3,4,5)P₃]. Phosphoinositide lipids with different phosphorylation states can recruit specific cytoplasmic effectors to trigger diverse cellular events, including cytoskeletal reorganization, ion channel regulation, and organelle trafficking (12). In particular, interactions between phosphoinositide lipids and bin/amphiphysin/rvs (BAR)-domain proteins can stimulate membrane curvature modulation and receptor endocytosis (13, 14). The conversions between phosphoinositide lipids may involve multiple steps and are dynamically regulated by site-specific kinases and phosphatases (15). In particular, the production of PI(3,4)P₂ can be controlled by two different pathways (16). Dephosphorylation of PI(3,4,5)P₃ by phosphoinositide 5-phosphatases can result in the production of PI(3,4)P₂ (17). On the other hand, class II PI3Ks can directly phosphorylate PI(4)P and contribute to PI(3,4)P₂ production (18).In the invadopodium, PI(3,4,5)P₃ and PI(3,4)P₂ act as important upstream messengers to initiate F-actin polymerization (3). However, the functional role of phosphoinositide lipids in adhesion turnover and integrin trafficking at invadopodia remains unclear. Here, we use the invadopodium model system of Src-transformed fibroblast (MEF-Src) (3, 19, 20) and MDA-MB-231 breast cancer cell to reveal how the PI(3,4)P₂ production and the microtubule-mediated retrograde transport trigger the membrane tubulation, stimulate the integrin endocytosis, and promote the invasive cell migration.     

Viewing rare conformations of the β2 adrenergic receptor with pressure-resolved DEER spectroscopy

The β₂ adrenergic receptor (β₂AR) is an archetypal G protein coupled receptor (GPCR). One structural signature of GPCR activation is a large-scale movement (ca. 6 to 14 Å) of transmembrane helix 6 (TM6) to a conformation which binds and activates a cognate G protein. The β₂AR exhibits a low level of agonist-independent G protein activation. The structural origin of this basal activity and its suppression by inverse agonists is unknown but could involve a unique receptor conformation that promotes G protein activation. Alternatively, a conformational selection model proposes that a minor population of the canonical active receptor conformation exists in equilibrium with inactive forms, thus giving rise to basal activity of the ligand-free receptor. Previous spin-labeling and fluorescence resonance energy transfer experiments designed to monitor the positional distribution of TM6 did not detect the presence of the active conformation of ligand-free β₂AR. Here we employ spin-labeling and pressure-resolved double electron–electron resonance spectroscopy to reveal the presence of a minor population of unliganded receptor, with the signature outward TM6 displacement, in equilibrium with inactive conformations. Binding of inverse agonists suppresses this population. These results provide direct structural evidence in favor of a conformational selection model for basal activity in β₂AR and provide a mechanism for inverse agonism. In addition, they emphasize 1) the importance of minor populations in GPCR catalytic function; 2) the use of spin-labeling and variable-pressure electron paramagnetic resonance to reveal them in a membrane protein; and 3) the quantitative evaluation of their thermodynamic properties relative to the inactive forms, including free energy, partial molar volume, and compressibility.

Many aspects of physiology in health and disease are regulated by signal transduction through G protein coupled receptors (GPCRs). Among these, the β₂ adrenergic receptor (β₂AR) is an archetype for the subset of family A GPCRs activated by hormones and neurotransmitters, as well as a pharmaceutical target for asthma and chronic obstructive pulmonary disease. The activity of β₂AR and other ligand-binding GPCRs can be finely tuned by ligands of varying efficacy, with agonists stimulating an increase in activation of cognate G proteins and inverse agonists decreasing G protein activation below a basal level.β₂AR crystal structures (1) have defined the outward movement of transmembrane helix 6 (TM6) as the largest structural rearrangement associated with activation of the receptor, as was originally found for rhodopsin (2) and subsequently for other receptors. This movement of TM6 is required for β₂AR to productively couple to its signaling partners (3).Ligand-independent or basal activity has been observed in many GPCRs. The level of basal receptor activity is highly receptor-specific and is important in maintaining homeostasis in physiologic systems independent of agonist stimulation (4). Basal activity in the β₂AR (5–7) and other receptors (4, 8) could arise from distinct receptor conformations that promote weak activation of the G protein, possibly involving an induced fit mechanism for receptor–G protein interaction. Alternatively, the basal activity could arise from a preexisting equilibrium (9, 10) between inactive and active conformations, where a small fraction of active receptors could account for baseline levels of signaling in physiologic systems. In support of this mechanism, sparse NMR data on the unliganded adenosine receptor provides evidence for the existence of an equilibrium between the inactive and active conformations (11).Although data from single-molecule fluorescence on β₂AR (12) have been interpreted in terms of the presence of the active form of the unliganded receptor, direct structural evidence for the existence of an active-like conformation in equilibrium with the unliganded, inactive conformation is lacking for this pharmacologically important GPCR. Indeed, structural studies of equilibrium populations by single-molecule fluorescence resonance energy transfer (FRET) (13) and double electron–electron resonance (DEER) spectroscopy (5) did not detect the presence of the active conformation in the ensemble of the unliganded β₂AR. This leaves the mechanism of basal activity for β₂AR unresolved. If true conformational selection plays a role, then a population of active form must exist in the equilibrium manifold of the unliganded receptor. The goal of the present work is to investigate the possibility that an active conformation in fact exists but is too sparsely populated to detect with the methods employed.Detection and characterization of sparsely populated (rare or excited) conformations is a general problem in elucidating the molecular mechanisms of protein function. Such low-lying excited states may play important functional roles despite being sparsely populated (14). Extensive empirical evidence indicates that the application of pressure may provide a solution to this problem by reversibly increasing the population of excited states (15, 16), allowing the use of standard experimental techniques to characterize these states and elucidate their functional roles. The mechanisms underlying the pressure-dependent conformational shifts are discussed in recent literature (17–20). It is important to note that as long as the pressure-induced shift in population is reversible, the rare state must also exist at atmospheric pressure and is not an artifact of pressure itself.Recently, site-directed spin labeling (SDSL) together with variable pressure electron paramagnetic resonance (EPR) was developed to monitor protein conformational shifts due to applied hydrostatic pressure (21). Of particular interest for the present study is SDSL with DEER (22). DEER provides a probability distribution of the distances between site-specifically introduced spin labels. Thus, each conformation in an ensemble generates a different distance in the distribution with a probability proportional to the population of the associated conformation, provided that the spin labels are properly placed to monitor the conformational equilibrium. In this respect, DEER is well suited to provide a structural definition to excited states revealed by pressure because it does not require a single conformation to be fully populated but instead reveals each conformation in the ensemble that is sufficiently populated (above ∼5 to 10%). Here we employ pressure-resolved DEER (23) to identify and characterize sparsely populated states (below 1%) in the conformational ensemble of unliganded β₂AR. The data provide direct structural evidence for the presence of an active-like conformation in the equilibrium ensemble of the unliganded receptor. The addition of agonists and inverse agonists increase and decrease the equilibrium population of the active form, respectively. The results indicate a mechanism for basal activity as well as that for inverse agonists in the β₂AR. Equally important, the variable pressure data provide a thermodynamic characterization of the active state including the partial molar volume, free energy, and compressibility relative to the ground (inactive) state.     

TMEM16C is involved in thermoregulation and protects rodent pups from febrile seizures

Febrile seizures (FSs) are the most common convulsion in infancy and childhood. Considering the limitations of current treatments, it is important to examine the mechanistic cause of FSs. Prompted by a genome-wide association study identifying TMEM16C (also known as ANO3) as a risk factor of FSs, we showed previously that loss of TMEM16C function causes hippocampal neuronal hyperexcitability [Feenstra et al., Nat. Genet. 46, 1274–1282 (2014)]. Our previous study further revealed a reduction in the number of warm-sensitive neurons that increase their action potential firing rate with rising temperature of the brain region harboring these hypothalamic neurons. Whereas central neuronal hyperexcitability has been implicated in FSs, it is unclear whether the maximal temperature reached during fever or the rate of body temperature rise affects FSs. Here we report that mutant rodent pups with TMEM16C eliminated from all or a subset of their central neurons serve as FS models with deficient thermoregulation. Tmem16c knockout (KO) rat pups at postnatal day 10 (P10) are more susceptible to hyperthermia-induced seizures. Moreover, they display a more rapid rise of body temperature upon heat exposure. In addition, conditional knockout (cKO) mouse pups (P11) with TMEM16C deletion from the brain display greater susceptibility of hyperthermia-induced seizures as well as deficiency in thermoregulation. We also found similar phenotypes in P11 cKO mouse pups with TMEM16C deletion from Ptgds-expressing cells, including temperature-sensitive neurons in the preoptic area (POA) of the anterior hypothalamus, the brain region that controls body temperature. These findings suggest that homeostatic thermoregulation plays an important role in FSs.

Body temperature is a critical index of health in mammals, while fever is an evolutionarily conserved response to infection (1). Febrile seizures (FSs), a convulsion triggered by fever in infants and young children ranging from 6 mo to 5 y of age (2–4), are the most common type of convulsion in youth. From 2 to 5% of American children display FSs in their first 5 y of life. Of those who had a first febrile seizure, 40% experienced recurrences of seizures (5). Whether FS genesis is associated with the rate of the core temperature (T_c) increase and/or when the T_c crosses a certain threshold for FS onset is unclear (6, 7). This question is difficult to address in clinical studies because of the paucity of information regarding the rate of T_c rise prior to a febrile seizure and the T_c at the onset of seizures. Genetic animal models that simulate the syndrome of FSs will greatly facilitate our understanding of FS susceptibility, beyond the known involvement of central neuronal hyperexcitability (8–12). A panoramic view of the pathogenesis of FSs enables future efforts to develop preventive strategies and potential treatments beyond currently available options (13, 14).A genome-wide association study (GWAS) of Danish children with or without a history of FSs in their first 2 y of life identified four loci as genetic risk factors, with TMEM16C (ANO3) showing the most significant association with greater FS susceptibility (15). Whereas this variant within intron 1 of TMEM16C (15) nearly doubles the risk for febrile seizures (3, 16, 17), whether and how TMEM16C gene activity may be involved in febrile seizures remains an open question. TMEM16C belongs to the TMEM16 family that includes Ca²⁺-activated Cl⁻ channels and Ca²⁺-activated scramblases (18, 19). In rat dorsal root ganglia (DRG), TMEM16C enhances the activity of a Na⁺-activated K⁺ channel thereby modulating pain sensitivity (20). Moreover, TMEM16C mutations are found in patients with dominant craniocervical dystonia, with their fibroblasts displaying a reduction in the thapsigargin-sensitive Ca²⁺ pool in the endoplasmic reticulum (21). Multiple missense mutations of TMEM16C have been associated with dominant dystonia (22).To examine the contribution of TMEM16C to FS susceptibility, we tested rat pups with or without TMEM16C (20) for their susceptibility to hyperthermia-induced seizures, an experimental model that simulates febrile seizures (23, 24). We also generated conditional knockout (cKO) mice bearing floxed alleles of Tmem16c (Ano3) for the purpose of removing TMEM16C via Cre recombinase. By conducting assessment of seizure-like behavior concurrent with T_c monitoring of rodent pups upon exposure to heat, we quantified the extent of T_c rise leading to the onset of seizures, thereby revealing that mutant pups exhibited greater susceptibility to hyperthermia-induced seizures as well as more rapid increases in T_c upon heat exposure.In mammals, thermoregulation is mediated by the preoptic area of the anterior hypothalamus (POA), involving “temperature-sensitive neurons” in this region that respond to brain temperature change with alteration of spontaneous action potential firing frequency (25–29). Intrigued by the drastic reduction of warm-sensitive POA neurons in rat pups lacking TMEM16C compared to littermate wild-type (WT) controls (15), we identified the Ptgds gene as a marker for temperature-sensitive POA neurons (30). Using the mouse line of PGDS-Cre (31), we examined cKO mouse pups with TMEM16C eliminated from temperature-sensitive POA neurons. Our findings that these cKO mutant pups showed not only deficient thermoregulation but also greater susceptibility to hyperthermia-induced seizures raise the possibility that compromised thermoregulation contributes to FS genesis.     

Anomalous mechanics of Zn2+-modified fibrin networks

Fibrin is the main component of blood clots. The mechanical properties of fibrin are therefore of critical importance in successful hemostasis. One of the divalent cations released by platelets during hemostasis is Zn²⁺; however, its effect on the network structure of fibrin gels and on the resultant mechanical properties remains poorly understood. Here, by combining mechanical measurements with three-dimensional confocal microscopy imaging, we show that Zn²⁺ can tune the fibrin network structure and alter its mechanical properties. In the presence of Zn²⁺, fibrin protofibrils form large bundles that cause a coarsening of the fibrin network due to an increase in fiber diameter and reduction of the total fiber length. We further show that the protofibrils in these bundles are loosely coupled to one another, which results in a decrease of the elastic modulus with increasing Zn²⁺ concentrations. We explore the elastic properties of these networks at both low and high stress: At low stress, the elasticity originates from pulling the thermal slack out of the network, and this is consistent with the thermal bending of the fibers. By contrast, at high stress, the elasticity exhibits a common master curve consistent with the stretching of individual protofibrils. These results show that the mechanics of a fibrin network are closely correlated with its microscopic structure and inform our understanding of the structure and physical mechanisms leading to defective or excessive clot stiffness.

Fibrin is the major component of blood clots, which stops bleeding from wound sites of blood vessels (1, 2). Upon injury, blood clots form when fibrinogen is converted to fibrin monomers, which polymerize into a fibrous gel that can withstand the pressure from the flowing blood and can therefore stop further blood loss (3–6). The mechanical properties of fibrin gels determine the performance of blood clots during hemostasis (7, 8): They must be mechanically strong enough to withstand the pressure of arterial blood; otherwise, the clots will not stop the loss of blood (9). They must also be strong enough to withstand the viscous forces; otherwise, parts of the gel may break off and be carried in the blood, where they may lodge in a vessel in the brain or the heart, which can cause a stroke or a heart attack (10, 11). The pressure of the blood is not constant; instead, it varies over a wide range, depending on locations in the body (12). Thus, the mechanical response of fibrin gels to the extent of pressure, or stress, is also crucial in determining the success of hemostasis. Fibrin gels exhibit a stress-dependent mechanical response (13, 14), similar to the gel networks formed from many other biopolymers, including actin, vimentin, neurofilaments, and collagen (15). Under small stresses, fibrin gels exhibit linear elasticity with the applied stress linearly proportional to the strain. By contrast, under large stresses, fibrin gels exhibit stress stiffening with the applied stress increasing nonlinearly with the strain.The mechanical property of the fibrin gel depends on the concentration of fibrinogen, as well as on many other factors (16–20). For example, the stiffness increases with the concentration of divalent cations such as Ca²⁺, which effectively acts as an additional cross-linker leading to the formation of a network from the filaments (17). Intriguingly, however, another divalent cation, Zn²⁺, seems to have the opposite effect: The stiffness of clots decreases with increasing concentration of Zn²⁺ (17); furthermore, the permeability of clots increases with increasing concentration of Zn²⁺ (21). This has important consequences as Zn²⁺, the second most abundant trace metal ion in the body (22, 23), is released from activated platelets during hemostasis, which can locally change its concentration (18, 19). Furthermore, Zn²⁺ deficiency in the blood is associated with abnormal blood clotting (24, 25). Nevertheless, the origin of effects and the impact of Zn²⁺ on the structure and properties of blood clots remain unclear. The effect of the addition of Zn²⁺ is correlated with the formation of a sparser network in the fibrin gel, as observed with two-dimensional (2D) scanning electron microscopy (SEM) (21). However, the observed network morphology is likely altered by drying during sample preparation (26), and 2D images cannot provide complete information about the network morphology; thus, the effects of the addition of Zn²⁺ on the three-dimensional (3D) structure of the gel network remains unknown. To understand the origin of the unusual decrease in stiffness upon addition of Zn²⁺, the mechanics of the fibrin gel must be correlated with Zn²⁺-induced changes in its network structure and properties.In this paper, we correlate the 3D structure of fibrin networks formed in the presence of Zn²⁺ with their mechanical properties to determine the consequences of the structure on the mechanical properties of fibrin gels. We use confocal microscopy to probe the 3D structure of the gel in its hydrated state and rheological measurements to probe its mechanics. We focus on the fully gelled structure, where the network has reached its steady state; thus, we can measure the confocal microscopy and rheology on separate samples whose structure and properties will nevertheless be identical. We find that as the Zn²⁺ concentration increases, the diameter of the fibrin fibers in the gel becomes measurably thicker while the total length of the fibrin fibers in the network becomes shorter; these results are explained by an increase in the number of protofibrils that are bundled together to form each fiber in the network. Bulk rheological measurements of the small-stress, linear elastic modulus of these gels are consistent with this structural packing of the protofibrils in the fibers; moreover, these results show that the protofibrils are not strongly coupled to one another in the fibers, which explains why the network becomes softer as the concentration of Zn²⁺ increases. At intermediate applied stresses, pronounced stress-stiffening is observed. Remarkably, at large applied stresses, the data from all the networks can be scaled together, indicating that the elastic modulus of the fibrin gels results from stretching of the individual fibrin protofibril that forms the bundles that make up the network. These results show that the mechanics of a fibrin network is correlated with its microscopic structure and provide important insight into the effect of Zn²⁺ on the mechanics of blood clots.     

Modulating the voltage sensor of a cardiac potassium channel shows antiarrhythmic effects

Cardiac arrhythmias are the most common cause of sudden cardiac death worldwide. Lengthening the ventricular action potential duration (APD), either congenitally or via pathologic or pharmacologic means, predisposes to a life-threatening ventricular arrhythmia, Torsade de Pointes. I_Ks (KCNQ1+KCNE1), a slowly activating K⁺ current, plays a role in action potential repolarization. In this study, we screened a chemical library in silico by docking compounds to the voltage-sensing domain (VSD) of the I_Ks channel. Here, we show that C28 specifically shifted I_Ks VSD activation in ventricle to more negative voltages and reversed the drug-induced lengthening of APD. At the same dosage, C28 did not cause significant changes of the normal APD in either ventricle or atrium. This study provides evidence in support of a computational prediction of I_Ks VSD activation as a potential therapeutic approach for all forms of APD prolongation. This outcome could expand the therapeutic efficacy of a myriad of currently approved drugs that may trigger arrhythmias.

I_Ks (KCNQ1+KCNE1), a slowly activating delayer rectifier in the heart, is important in controlling cardiac action potential duration (APD) and adaptation of heart rate in various physiological conditions (1). The I_Ks potassium channel has slow activation kinetics, and the activation terminates cardiac action potentials (APs) (2). This channel is formed by the voltage-gated potassium (K_V) channel subunit KCNQ1 and the regulatory subunit KCNE1. The association of KCNE1 drastically alters the phenotype of the channel, including a shift of voltage dependence of activation to more positive voltages, a slower activation time course, a changed ion selectivity, and different responses to drugs and modulators (3–6). Similar to other K_V channels, KCNQ1 has six transmembrane segments, S1 to S6, in which S1 to S4 form the voltage-sensing domain (VSD), while S5 and S6 form the pore domain (PD); four KCNQ1 subunits comprise the KCNQ1 channel (7, 8). KCNQ1 and I_Ks channels are activated by voltage. The VSD in response to membrane depolarization changes conformation, triggered by the movements of the S4 segment that contains positively charged residues (9–15). This conformational change alters the interactions between the VSD and the pore, known as the VSD–pore coupling, to induce pore opening (12–15).The ventricular APD depends on the balance of outward and inward currents flowing at plateau potentials. The outward currents include the delayed rectifiers I_Kr and I_Ks, while the inward currents include a persistent sodium current (I_NaP) (16). Specific mutations in any of these channel proteins that cause a reduction in outward current or increase in inward current are associated with congenital long QT syndrome (LQTS). The QT interval is the time between the initial depolarization of the ventricle until the time to full repolarization. LQTS is a condition in which the APD is abnormally prolonged, predisposing the afflicted patients to a lethal cardiac arrhythmia called Torsades de Pointes (TdP) (17). In fact, mutations in multiple genes that alter the function of various ion channels have been associated with LQTS (18). There is also a much more prevalent problem called acquired LQTS (aLQTS) that is most often associated with off target effects of drugs. Many drugs are marketed with a QT prolongation warning, and the drug concentrations that can be used therapeutically are limited by this potentially lethal side effect. Some effective drugs have been removed from the market (19) because of QT prolongation, and others are abandoned before clinical trials even began. Therefore, aLQTS is costly for the pharmaceutical industry both in drug development (to avoid this side effect) and when it results in removal from the market of compounds that have effectively treated other diseases (20, 21). At present, the I_Kr (HERG) potassium channel (20) and the phosphoinositide 3-kinase (PI3K) (22, 23) have been identified as the most prominent off targets of these drugs for the association with aLQTS.We hypothesized that in LQTS, the normal heart function can be restored and QT prolongation prevented by compensating for the change in net current from any of the channels produced by the myocyte; all that is required is that a reasonable facsimile of normal net current flow be restored. We applied this approach to aLQTS based on a computational study to show that a shift of voltage-dependent activation of I_Ks to more negative voltages would increase I_Ks during ventricular APs; this increase of I_Ks would revert drug-induced APD prolongation to normal. More importantly, a change in I_Ks voltage-dependent activation might affect the normal APD to a much smaller degree because of its slow activation kinetics in healthy ventricular myocytes, thereby posing minimal risk of cardiac toxicity on its own. To apply this approach experimentally, we needed a compound that could specifically shift the voltage dependence of I_Ks activation. Previous studies showed that the benzodiazepine R-L3 (24) and polyunsaturated fatty acids (25) activate I_Ks channels, while more recently, rottlerin was shown to act similarly to R-L3 (26). However, the effects of these compounds on I_Ks are complex, likely through binding to more than one site in the channel protein instead of simply acting on voltage-dependent activation (24, 27, 28). In addition, these compounds showed poor specificity for I_Ks, also affecting other ion channels in the heart (27, 29, 30), which make these compounds unsuitable to test our hypothesis. Recently, we have identified CP1 as an activator for I_Ks, which mimics the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) to mediate the VSD–pore coupling (13, 31). CP1 enhances I_Ks primarily by increasing current amplitude with some shift of voltage dependence of activation, which is not suitable for our test either.In this study, we identified a compound, C28, using an approach that combines in silico and experimental screening, that interacts with the KCNQ1 VSD and shifts voltage dependence of VSD activation to more negative voltages. C28 increases both exogenously expressed I_Ks and the current in native cardiac myocytes. As predicted by computational modeling, C28 can prevent or reverse the drug-induced APD prolongation back to normal while having a minimal effect on the control APD at the same concentration in healthy cardiac myocytes. This study demonstrates that the KCNQ1 VSD can be used as a drug target for developing a therapy for LQTS, and C28 identified in this study may be used as a lead for this development. Furthermore, our results provide support for the use of docking computations based on ion channel structure and cellular physiology, in combination with functional studies based on molecular mechanisms, as an effective approach for rational drug design.     

Regulation of longevity by depolarization-induced activation of PLC-β–IP3R signaling in neurons

Mitochondrial ATP production is a well-known regulator of neuronal excitability. The reciprocal influence of plasma-membrane potential on ATP production, however, remains poorly understood. Here, we describe a mechanism by which depolarized neurons elevate the somatic ATP/ADP ratio in Drosophila glutamatergic neurons. We show that depolarization increased phospholipase-Cβ (PLC-β) activity by promoting the association of the enzyme with its phosphoinositide substrate. Augmented PLC-β activity led to greater release of endoplasmic reticulum Ca²⁺ via the inositol trisphosphate receptor (IP₃R), increased mitochondrial Ca²⁺ uptake, and promoted ATP synthesis. Perturbations that decoupled membrane potential from this mode of ATP synthesis led to untrammeled PLC-β–IP₃R activation and a dramatic shortening of Drosophila lifespan. Upon investigating the underlying mechanisms, we found that increased sequestration of Ca²⁺ into endolysosomes was an intermediary in the regulation of lifespan by IP₃Rs. Manipulations that either lowered PLC-β/IP₃R abundance or attenuated endolysosomal Ca²⁺ overload restored animal longevity. Collectively, our findings demonstrate that depolarization-dependent regulation of PLC-β–IP₃R signaling is required for modulation of the ATP/ADP ratio in healthy glutamatergic neurons, whereas hyperactivation of this axis in chronically depolarized glutamatergic neurons shortens animal lifespan by promoting endolysosomal Ca²⁺ overload.

Spatially circumscribed ATP production at nerve termini is predicated on local mitochondria that are energized when voltage-gated Ca²⁺ channels provide the [Ca²⁺] elevations needed to overcome the low sensitivity of the mitochondrial Ca²⁺ uniporter (MCU) (1–3). In neuronal soma, however, bulk cytosolic [Ca²⁺] is not elevated to levels needed for mitochondrial sequestration. Rather, mitochondrial Ca²⁺ uptake in the somatodendritic compartment occurs at specialized points of contact between mitochondria and endoplasmic reticulum (ER) where Ca²⁺ released by IP₃Rs is transferred into the mitochondrial matrix (4). Approximately 75 to 90% of the somatic ATP synthesized following interorganellar transfer of Ca²⁺ is consumed by Na⁺/K⁺ ATPases, which help establish resting membrane potential and permit repolarization during activity (5, 6). Therefore, defects in neuronal ATP synthesis result in loss of membrane potential and hyperexcitability (6).Whether excitability of the somatic plasma membrane (PM) exerts reciprocal influence on mitochondrial [Ca²⁺] and ATP production remains poorly understood. In an attempt to fill some of the gaps in knowledge, we examined the effects of PM potential on mitochondrial ATP production and Ca²⁺ homeostasis in Drosophila neurons. Owing to recent reports of neuronal hyperexcitability being a driver of diminished longevity in organisms ranging from Caenorhabditis elegans to humans (7–9), we hoped our studies would inform insights into the regulation of aging and lifespan. Moreover, since neuronal hyperexcitability, Ca²⁺ dyshomeostasis, and bioenergetic dysfunction characterize neurodegenerative diseases (6, 10, 11), uncovering actionable molecular targets that bridge these perturbations may bear therapeutic value. Our findings reveal a previously unknown mechanism by which excitability regulates bioenergetics and Ca²⁺ signaling and points to the utility of this signaling circuit in the regulation of longevity.     

The relationship between birth timing,circuit wiring,and physiological response properties of cerebellar granule cells

Cerebellar granule cells (GrCs) are usually regarded as a uniform cell type that collectively expands the coding space of the cerebellum by integrating diverse combinations of mossy fiber inputs. Accordingly, stable molecularly or physiologically defined GrC subtypes within a single cerebellar region have not been reported. The only known cellular property that distinguishes otherwise homogeneous GrCs is the correspondence between GrC birth timing and the depth of the molecular layer to which their axons project. To determine the role birth timing plays in GrC wiring and function, we developed genetic strategies to access early- and late-born GrCs. We initiated retrograde monosynaptic rabies virus tracing from control (birth timing unrestricted), early-born, and late-born GrCs, revealing the different patterns of mossy fiber input to GrCs in vermis lobule 6 and simplex, as well as to early- and late-born GrCs of vermis lobule 6: sensory and motor nuclei provide more input to early-born GrCs, while basal pontine and cerebellar nuclei provide more input to late-born GrCs. In vivo multidepth two-photon Ca²⁺ imaging of axons of early- and late-born GrCs revealed representations of diverse task variables and stimuli by both populations, with modest differences in the proportions encoding movement, reward anticipation, and reward consumption. Our results suggest neither organized parallel processing nor completely random organization of mossy fiber→GrC circuitry but instead a moderate influence of birth timing on GrC wiring and encoding. Our imaging data also provide evidence that GrCs can represent generalized responses to aversive stimuli, in addition to recently described reward representations.

Cerebellar granule cells (GrCs) comprise the majority of neurons in the mammalian brain (1, 2). Each GrC receives only four excitatory inputs from mossy fibers, which originate in a variety of brainstem nuclei and the spinal cord, and the vast number of GrCs permits diverse combinations of mossy fiber inputs. Classical theories of cerebellar function have therefore proposed that GrCs integrate diverse, multimodal mossy fiber inputs and thus collectively expand coding space in the cerebellum (3–5). Until recently, studies have focused on the role of GrCs in implementing sparse coding of sensorimotor variables and stimuli (6–9). However, recent physiological studies of GrCs in awake, behaving animals highlight GrC encoding of cognitive signals in addition to sensorimotor signals (10–13). GrCs have also been recently shown to encode denser representations than expected by classical theory (10–12, 14–18), including a lack of dimensionality expansion under certain conditions (18).Despite the vast number of GrCs, stable molecularly or physiologically defined GrC subtypes within a single cerebellar region or lobule have not been described (19–22), although variation in gene expression across different regions has been reported (22, 23). The only known axis along which spatially intermingled GrCs can be distinguished from each other is the depth of the molecular layer to which their parallel fiber axons (PFs) project, which is dictated by GrC lineage and birth timing (24, 25). Birth timing predicts the wiring and functional properties of diverse neuron types in many neural systems (26), including the neocortex (27, 28), other forebrain regions (29, 30), olfactory bulb (31–33), and ventral spinal cord (34, 35). Furthermore, classic studies utilizing γ-irradiation at different times during rat postnatal development to ablate different cerebellar GrC and interneuron populations suggested that GrCs born at different times could contribute differentially to motor vs. action coordination (36). These observations also led to an as-of-yet untested hypothesis that mossy fibers arriving at different times during development could connect with different GrC populations. Could GrC birth timing be an organizing principle for information processing in the cerebellum?Recent evidence and modeling point to the possibility of spatial clusters of coactivated PFs (15, 37), suggesting that GrCs born around the same time may disproportionally receive coactive mossy fiber inputs. However, another study using different methods and stimuli did not find differences in the physiological responses of early- and late-born GrCs to various sensorimotor stimuli (38). Here, we address the role of birth timing in GrC wiring and function. We developed strategies to gain genetic access to early- and late-born GrCs, as well as control GrCs not restricted by birth timing. We report the first monosynaptic input tracing to GrCs, finding differential mossy fiber inputs to GrCs in vermis lobule 6 and simplex, as well as different patterns of input to early- and late-born GrCs in vermis lobule 6. Finally, we performed in vivo multidepth two-photon Ca²⁺ imaging of PFs of early- and late-born GrCs during an operant task and presentation of a panel of sensory, appetitive, and aversive stimuli. We found modest differences in the proportions of early- and late-born GrCs encoding of a subset of movement and reward parameters. Together, these results reveal a contribution of GrC birth timing to their input wiring and diverse encoding properties.