Parathyroid hormone initiates dynamic NHERF1 phosphorylation cycling and conformational changes that regulate NPT2A-dependent phosphate transport.

Na+-H+ exchanger regulatory factor-1 (NHERF1) is a PDZ protein that scaffolds membrane proteins, including sodium-phosphate co-transport protein 2A (NPT2A) at the plasma membrane. NHERF1 is a phosphoprotein with 40 Ser and Thr residues. Here, using tandem MS analysis, we characterized the sites of parathyroid hormone (PTH)-induced NHERF1 phosphorylation and identified 10 high-confidence phosphorylation sites. Ala replacement at Ser46, Ser162, Ser181, Ser269, Ser280, Ser291, Thr293, Ser299, and Ser302 did not affect phosphate uptake, but S290A substitution abolished PTH-dependent phosphate transport. Unexpectedly, Ser290 was rapidly dephosphorylated and rephosphorylated after PTH stimulation, and we found that protein phosphatase 1α (PP1α), which binds NHERF1 through a conserved VxF/W PP1 motif, dephosphorylates Ser290 Mutating 257VPF259 eliminated PP1 binding and blunted dephosphorylation. Tautomycetin blocked PP1 activity and abrogated PTH-sensitive phosphate transport. Using fluorescence lifetime imaging (FLIM), we observed that PTH paradoxically and transiently elevates intracellular phosphate. Added phosphate blocked PP1α-mediated Ser290 dephosphorylation of recombinant NHERF1. Hydrogen-deuterium exchange MS revealed that ß-sheets in NHERF1's PDZ2 domain display lower deuterium uptake than those in the structurally similar PDZ1, implying that PDZ1 is more cloistered. Dephosphorylated NHERF1 exhibited faster exchange at C-terminal residues suggesting that NHERF1 dephosphorylation precedes Ser290 rephosphorylation. Our results show that PP1α and NHERF1 form a holoenzyme and that a multiprotein kinase cascade involving G protein-coupled receptor kinase 6A controls the Ser290 phosphorylation status of NHERF1 and regulates PTH-sensitive, NPT2A-mediated phosphate uptake. These findings reveal how reversible phosphorylation modifies protein conformation and function and the biochemical mechanisms underlying PTH control of phosphate transport.

Site-specific polyubiquitination differentially regulates parathyroid hormone receptor-initiated MAPK signaling and cell proliferation.

G protein-coupled receptor (GPCR) signaling and trafficking are essential for cellular function and regulated by phosphorylation, ß-arrestin, and ubiquitination. The GPCR parathyroid hormone receptor (PTHR) exhibits time-dependent reversible ubiquitination. The exact ubiquitination sites in PTHR are unknown, but they extend upstream of its intracellular tail. Here, using tandem MS, we identified Lys388 in the third loop and Lys484 in the C-terminal tail as primary ubiquitination sites in PTHR. We found that PTHR ubiquitination requires ß-arrestin and does not display a preference for ß-arrestin1 or -2. PTH stimulated PTHR phosphorylation at Thr387/Thr392 and within the Ser489-Ser493 region. Such phosphorylation events may recruit ß-arrestin, and we observed that chemically or genetically blocking PTHR phosphorylation inhibits its ubiquitination. Specifically, Ala replacement at Thr387/Thr392 suppressed ß-arrestin binding and inhibited PTHR ubiquitination, suggesting that PTHR phosphorylation and ubiquitination are interdependent. Of note, Lys-deficient PTHR mutants promoted normal cAMP formation, but exhibited differential mitogen-activated protein kinase (MAPK) signaling. Lys-deficient PTHR triggered early onset and delayed ERK1/2 signaling compared with wildtype PTHR. Moreover, ubiquitination of Lys388 and Lys484 in wildtype PTHR strongly decreased p38 signaling, whereas Lys-deficient PTHR retained signaling comparable to unstimulated wildtype PTHR. Lys-deficient, ubiquitination-refractory PTHR reduced cell proliferation and increased apoptosis. However, elimination of all 11 Lys residues in PTHR did not affect its internalization and recycling. These results pinpoint the ubiquitinated Lys residues in PTHR controlling MAPK signaling and cell proliferation and survival. Our findings suggest new opportunities for targeting PTHR ubiquitination to regulate MAPK signaling or manage PTHR-related disorders.

Growing season moisture drives interannual variation in woody productivity of a temperate deciduous forest.

The climate sensitivity of forest ecosystem woody productivity (ANPPstem ) influences carbon cycle responses to climate change. For the first time, we combined long-term annual growth and forest census data of a diverse temperate broadleaf deciduous forest, seeking to resolve whether ANPPstem is primarily moisture- or energy-limited and whether climate sensitivity has changed in recent decades characterised by more mesic conditions and elevated CO2 . We analysed tree-ring chronologies across 109 yr of monthly climatic variation (1901-2009) for 14 species representing 97% of ANPPstem in a 25.6 ha plot in northern Virginia, USA. Radial growth of most species and ecosystem-level ANPPstem responded positively to cool, moist growing season conditions, but the same conditions in the previous May-July were associated with reduced growth. In recent decades (1980-2009), responses were more variable and, on average, weaker. Our results indicated that woody productivity is primarily limited by current growing season moisture, as opposed to temperature or sunlight, but additional complexity in climate sensitivity may reflect the use of stored carbohydrate reserves. Overall, while such forests currently display limited moisture sensitivity, their woody productivity is likely to decline under projected hotter and potentially drier growing season conditions.

Erythropoietin Induces Homeostatic Plasticity at Hippocampal Synapses.

The cytokine erythropoietin (EPO) is the master regulator of erythropoiesis. Intriguingly, many studies have shown that the cognitive performance of patients receiving EPO for its hematopoietic effects is enhanced, which prompted the growing interest in the use of EPO-based strategies to treat neuropsychiatric disorders. EPO plays key roles in brain development and maturation, but also modulates synaptic transmission. However, the mechanisms underlying the latter have remained elusive. Here, we show that acute (40-60 min) exposure to EPO presynaptically downregulates spontaneous and afferent-evoked excitatory transmission, without affecting basal firing of action potentials. Conversely, prolonged (3 h) exposure to EPO, if followed by a recovery period (1 h), is able to elicit a homeostatic increase in excitatory spontaneous, but not in evoked, synaptic transmission. These data lend support to the emerging view that segregated pathways underlie spontaneous and evoked neurotransmitter release. Furthermore, we show that prolonged exposure to EPO facilitates a form of hippocampal long-term potentiation that requires noncanonical recruitment of calcium-permeable AMPA receptors for its maintenance. These findings provide important new insight into the mechanisms by which EPO enhances neuronal function, learning, and memory.

ForC: a global database of forest carbon stocks and fluxes.

Forests play an influential role in the global carbon (C) cycle, storing roughly half of terrestrial C and annually exchanging with the atmosphere more than five times the carbon dioxide (CO2 ) emitted by anthropogenic activities. Yet, scaling up from field-based measurements of forest C stocks and fluxes to understand global scale C cycling and its climate sensitivity remains an important challenge. Tens of thousands of forest C measurements have been made, but these data have yet to be integrated into a single database that makes them accessible for integrated analyses. Here we present an open-access global Forest Carbon database (ForC) containing previously published records of field-based measurements of ecosystem-level C stocks and annual fluxes, along with disturbance history and methodological information. ForC expands upon the previously published tropical portion of this database, TropForC (https://doi.org/10.5061/dryad.t516f), now including 17,367 records (previously 3,568) representing 2,731 plots (previously 845) in 826 geographically distinct areas. The database covers all forested biogeographic and climate zones, represents forest stands of all ages, and currently includes data collected between 1934 and 2015. We expect that ForC will prove useful for macroecological analyses of forest C cycling, for evaluation of model predictions or remote sensing products, for quantifying the contribution of forests to the global C cycle, and for supporting international efforts to inventory forest carbon and greenhouse gas exchange. A dynamic version of ForC is maintained at on GitHub (https://GitHub.com/forc-db), and we encourage the research community to collaborate in updating, correcting, expanding, and utilizing this database. ForC is an open access database, and we encourage use of the data for scientific research and education purposes. Data may not be used for commercial purposes without written permission of the database PI. Any publications using ForC data should cite this publication and Anderson-Teixeira et al. (2016a) (see Metadata S1). No other copyright or cost restrictions are associated with the use of this data set.

Actin-Sorting Nexin 27 (SNX27)-Retromer Complex Mediates Rapid Parathyroid Hormone Receptor Recycling.

The G protein-coupled parathyroid hormone receptor (PTHR) regulates mineral-ion homeostasis and bone remodeling. Upon parathyroid hormone (PTH) stimulation, the PTHR internalizes into early endosomes and subsequently traffics to the retromer complex, a sorting platform on early endosomes that promotes recycling of surface receptors. The C terminus of the PTHR contains a type I PDZ ligand that binds PDZ domain-containing proteins. Mass spectrometry identified sorting nexin 27 (SNX27) in isolated endosomes as a PTHR binding partner. PTH treatment enriched endosomal PTHR. SNX27 contains a PDZ domain and serves as a cargo selector for the retromer complex. VPS26, VPS29, and VPS35 retromer subunits were isolated with PTHR in endosomes from cells stimulated with PTH. Molecular dynamics and protein binding studies establish that PTHR and SNX27 interactions depend on the PDZ recognition motif in PTHR and the PDZ domain of SNX27. Depletion of either SNX27 or VPS35 or actin depolymerization decreased the rate of PTHR recycling following agonist stimulation. Mutating the PDZ ligand of PTHR abolished the interaction with SNX27 but did not affect the overall rate of recycling, suggesting that PTHR may directly engage the retromer complex. Coimmunoprecipitation and overlay experiments show that both intact and mutated PTHR bind retromer through the VPS26 protomer and sequentially assemble a ternary complex with PTHR and SNX27. SNX27-independent recycling may involve N-ethylmaleimide-sensitive factor, which binds both PDZ intact and mutant PTHRs. We conclude that PTHR recycles rapidly through at least two pathways, one involving the ASRT complex of actin, SNX27, and retromer and another possibly involving N-ethylmaleimide-sensitive factor.

Carbon dynamics of mature and regrowth tropical forests derived from a pantropical database (TropForC-db).

Tropical forests play a critical role in the global carbon (C) cycle, storing ~45% of terrestrial C and constituting the largest component of the terrestrial C sink. Despite their central importance to the global C cycle, their ecosystem-level C cycles are not as well-characterized as those of extra-tropical forests, and knowledge gaps hamper efforts to quantify C budgets across the tropics and to model tropical forest-climate interactions. To advance understanding of C dynamics of pantropical forests, we compiled a new database, the Tropical Forest C database (TropForC-db), which contains data on ground-based measurements of ecosystem-level C stocks and annual fluxes along with disturbance history. This database currently contains 3568 records from 845 plots in 178 geographically distinct areas, making it the largest and most comprehensive database of its type. Using TropForC-db, we characterized C stocks and fluxes for young, intermediate-aged, and mature forests. Relative to existing C budgets of extra-tropical forests, mature tropical broadleaf evergreen forests had substantially higher gross primary productivity (GPP) and ecosystem respiration (Reco), their autotropic respiration (Ra) consumed a larger proportion (~67%) of GPP, and their woody stem growth (ANPPstem) represented a smaller proportion of net primary productivity (NPP, ~32%) or GPP (~9%). In regrowth stands, aboveground biomass increased rapidly during the first 20 years following stand-clearing disturbance, with slower accumulation following agriculture and in deciduous forests, and continued to accumulate at a slower pace in forests aged 20-100 years. Most other C stocks likewise increased with stand age, while potential to describe age trends in C fluxes was generally data-limited. We expect that TropForC-db will prove useful for model evaluation and for quantifying the contribution of forests to the global C cycle. The database version associated with this publication is archived in Dryad (DOI: 10.5061/dryad.t516f) and a dynamic version is maintained at https://github.com/forc-db.

Carbon storage in old-growth forests of the Mid-Atlantic: toward better understanding the eastern forest carbon sink.

Few old-growth stands remain in the matrix of secondary forests that dominates the eastern North American landscape. These remnant stands offer insight on the potential carbon (C) storage capacity of now-recovering secondary forests. We surveyed the remaining old-growth forests on sites characteristic of the general Mid-Atlantic United States and estimated the size of multiple components of forest C storage. Within and between old-growth stands, variability in C density is high and related to overstory tree species composition. The sites contain 219 ± 46 Mg C/ha (mean ± SD), including live and dead aboveground biomass, leaf litter, and the soil O horizon, with over 20% stored in downed wood and snags. Stands dominated by tulip poplar (Liriodendron tulipifera) store the most live biomass, while the mixed oak (Quercus spp.) stands overall store more dead wood. Total C density is 30% higher (154 Mg C/ha), and dead wood C density is 1800% higher (46 Mg C/ha) in the old-growth forests than in the surrounding younger forests (120 and 5 Mg C/ha, respectively). The high density of dead wood in old growth relative to secondary forests reflects a stark difference in historical land use and, possibly, the legacy of the local disturbance (e.g., disease) history. Our results demonstrate the potential for dead wood to maintain the sink capacity of secondary forests for many decades to come.

In vitro placenta barrier model using primary human trophoblasts, underlying connective tissue and vascular endothelium.

Fetal development may be compromised by adverse events at the placental interface between mother and fetus. However, it is still unclear how the communication between mother and fetus occurs through the placenta. In vitro - models of the human placental barrier, which could help our understanding and which recreate three-dimensional (3D) structures with biological functionalities and vasculatures, have not been reported yet. Here we present a 3D-vascularized human primary placental barrier model which can be constructed in 1 day. We illustrate the similarity of our model to first trimester human placenta, both in its structure and in its ability to respond to altered oxygen and to secrete factors that cause damage cells across the barrier including embryonic cortical neurons. We use this model to highlight the possibility that both the trophoblast and the endothelium within the placenta might play a role in the fetomaternal dialogue.

Cdc42 activation couples fluid shear stress to apical endocytosis in proximal tubule cells.

Cells lining the kidney proximal tubule (PT) respond to acute changes in glomerular filtration rate and the accompanying fluid shear stress (FSS) to regulate reabsorption of ions, glucose, and other filtered molecules and maintain glomerulotubular balance. Recently, we discovered that exposure of PT cells to FSS also stimulates an increase in apical endocytic capacity (Raghavan et al. PNAS, 111:8506-8511, 2014). We found that FSS triggered an increase in intracellular Ca2+ concentration ([Ca2+]i) that required release of extracellular ATP and the presence of primary cilia. In this study, we elucidate steps downstream of the increase in [Ca2+]i that link FSS-induced calcium increase to increased apical endocytic capacity. Using an intramolecular FRET probe, we show that activation of Cdc42 is a necessary step in the FSS-stimulated apical endocytosis cascade. Cdc42 activation requires the primary cilia and the FSS-mediated increase in [Ca2+]i Moreover, Cdc42 activity and FSS-stimulated endocytosis are coordinately modulated by activators and inhibitors of calmodulin. Together, these data suggest a mechanism by which PT cell exposure to FSS is translated into enhanced endocytic uptake of filtered molecules.

Secretions from placenta, after hypoxia/reoxygenation, can damage developing neurones of brain under experimental conditions.

Some psychiatric diseases in children and young adults are thought to originate from adverse exposures during foetal life, including hypoxia and hypoxia/reoxygenation. The mechanism is not understood. Several authors have emphasised that the placenta is likely to play an important role as the key interface between mother and foetus. Here we have explored whether a first trimester human placenta or model barrier of primary human cytotrophoblasts might secrete factors, in response to hypoxia or hypoxia/reoxygenation, that could damage neurones. We find that the secretions in conditioned media caused an increase of [Ca(2+)]i and mitochondrial free radicals and a decrease of dendritic lengths, branching complexity, spine density and synaptic activity in dissociated neurones from embryonic rat cerebral cortex. There was altered staining of glutamate and GABA receptors. We identify glutamate as an active factor within the conditioned media and demonstrate a specific release of glutamate from the placenta/cytotrophoblast barriers invitro after hypoxia or hypoxia/reoxygenation. Injection of conditioned media into developing brains of P4 rats reduced the numerical density of parvalbumin-containing neurones in cortex, hippocampus and reticular nucleus, reduced immunostaining of glutamate receptors and altered cellular turnover. These results show that the placenta is able to release factors, in response to altered oxygen, that can damage developing neurones under experimental conditions.

The small GTPase Arf1 modulates Arp2/3-mediated actin polymerization via PICK1 to regulate synaptic plasticity.

Inhibition of Arp2/3-mediated actin polymerization by PICK1 is a central mechanism to AMPA receptor (AMPAR) internalization and long-term depression (LTD), although the signaling pathways that modulate this process in response to NMDA receptor (NMDAR) activation are unknown. Here, we define a function for the GTPase Arf1 in this process. We show that Arf1-GTP binds PICK1 to limit PICK1-mediated inhibition of Arp2/3 activity. Expression of mutant Arf1 that does not bind PICK1 leads to reduced surface levels of GluA2-containing AMPARs and smaller spines in hippocampal neurons, which occludes subsequent NMDA-induced AMPAR internalization and spine shrinkage. In organotypic slices, NMDAR-dependent LTD of AMPAR excitatory postsynaptic currents is abolished in neurons expressing mutant Arf1. Furthermore, NMDAR stimulation downregulates Arf1 activation and binding to PICK1 via the Arf-GAP GIT1. This study defines Arf1 as a critical regulator of actin dynamics and synaptic function via modulation of PICK1.