Functional Analysis of PP1 Regulator Phactr1

The Phosphatase and actin regulator (Phactr) protein family has been identified as a group of four proteins (Phactr1,2,3,4), interacting with Protein Phosphatase 1 (PP1) and G-actin. G-actin binding to Phactr1 RPEL motifs has been shown to sterically inhibit Phactr1 interaction with PP1 and its nuclear import. Members of the Phactr family are highly expressed in the nervous system and have been implicated in the regulation of the actin cytoskeleton, cell migration and angiogenesis. The molecular mechanism of their signalling is however not well understood. Here I show that Phactr1 functions as a novel regulatory subunit of PP1. Phactr1 contains several binding motifs typical of PP1 regulatory subunits, including an RVxF motif, located in the C-terminal part of Phactr1, partially overlapping with the G-actin binding RPEL motif. Phactr1 binding leaves the PP1 active site accessible for substrates, blocks one of the substrate-binding grooves and creates a highly basic pocket along the PP1 hydrophobic substrate binding groove, thereby defining Phactr1-PP1 specific protein substrates for dephosphorylation. A phosphoproteomic analysis revealed that activation of Phactr1-PP1 complex formation leads to the dephosphorylation of several cytoskeletal proteins (such as IRSp53 and Afadin), leading to rearrangements of the actin cytoskeleton. Induction of Phactr1-PP1 interaction leads to the formation of aberrant actomyosin structures in fibroblasts. The converse phenotype could be induced by non-PP1-binding Phactr1 mutant expression, leading to the formation of long cytoplasmic extensions. In neurons, Phactr1 was enriched in dendritic spines and activation of Phactr1-PP1 complex formation led to changes in dendritic spine morphology. Moreover, I show that Phactr1 L519R mutation, that has been implicated in epilepsy, decreases Phactr1 affinity to G-actin, and thus indirectly activates Phactr1 interaction with PP1. All this suggests that Phactr1 targets PP1 to dephosphorylate specific cytoskeletal proteins, leading to changes in the actin cytoskeleton, which in neurons modulates dendritic spine morphology, leading to changes in neuronal signalling

δ15N systematics in two minerotrophic peatlands in the eastern U.S.: Insights into nitrogen cycling under moderate pollution

Availability of reactive nitrogen (Nr; mainly ammonium, NH4+, and nitrate, NO3−) via precipitation and upwelling groundwater affects carbon (C) accumulation in peat deposits and biological greenhouse gas emissions. Isotope composition of Nr was studied at Wolf Swamp (WS) and The Glades (GL), two peatlands located in western Maryland (U.S.), to provide a more robust understanding of N cycling at the ecosystem level. In recent years, both sites experienced a moderate atmospheric pollution (3–9 kg of deposited Nr ha−1 yr−1), and possible additional Nr inputs via groundwater contaminated by fertilizers/livestock. Sampling of living Sphagnum, vertical peat profiles, atmospheric deposition, and surface bog water was carried out on October 22–24, 2017. The δ15Nr signatures of vertical precipitation were negative (mass-weighted means of −7.2 and −8.1‰ at WS and GL, respectively) despite reports that NOx emitted by coal-burning power plants, a precursor of deposited nitrate, had been extremely positive (up to +26‰). Mean δ15N values at both sites increased in the order: vertical precipitation < living Sphagnum < atmospheric N2 < surface bog water < maturating peat. Intermediate δ15N values of living Sphagnum (−1.5‰ at both sites) suggested simultaneous incorporation of isotopically light atmospheric Nr and isotopically heavy Nr from bog water in the newly formed biomass. The δ15N values of bulk peat increased downcore by 4.5 and 4.9‰ at WS and GL, respectively. Gradual mineralization of organic N, mainly by nitrification and denitrification, is isotopically selective, leaving behind higher δ15N values of bulk peat at greater peat depths. Nitrogen concentration gradients and δ15N trends in vertical peat profiles at these locations therefore cannot be used as an archive of past changes in pollution levels and N isotope signatures of incoming Nr. Keywords: peat, Nitrogen, Isotopes, Atmospheric deposition, Diagenesi

Chromium isotope fractionations resulting from electroplating, chromating and anodizing: implications for groundwater pollution studies

Available online 27 March 2017A number of shallow aquifers in industrial regions have been polluted by toxic Cr(VI). At some sites, spontaneous reduction of dissolved Cr(VI) to insoluble Cr(III) has been observed. Precipitation of non-toxic Cr(III) is accompanied by a Cr isotope fractionation, with the residual Cr(VI) becoming enriched in the heavier isotope 53Cr, and depleted in the lighter isotope 52Cr. Thus far, δ53Cr values of the contamination source have been poorly constrained. These values are needed to quantify the extent of Cr(VI) reduction in the aquifers. We present δ53Cr values of solutions generated during Cr-electroplating, chromating and anodizing at nine industrial sites. The source chemical, CrO3, had a mean δ53Cr of 0.0‰. A small-to-negligible Cr isotope fractionation was observed between the solutions of the plating baths and the source chemical. Across all sample types, the mean δ53Cr(VI) value was 0.2‰. The mean δ53Cr(VI) value of contaminated groundwater in the same region, studied previously, was significantly higher (2.9‰), indicating Cr(VI) reduction. Based on low δ53Cr(VI) values of plating baths and rinsewaters as potential contamination sources, and their low variability, we suggest that most aquifer δ53Cr(VI) values higher than 1.0‰ are a result of in-situ Cr(VI) reduction.Martin Novak, Vladislav Chrastny, Ondrej Sebek, Eva Martinkova, Eva Prechova, Jan Curik, Frantisek Veselovsky, Marketa Stepanova, Barbora Dousova, Frantisek Buzek, Juraj Farkas, Alexandre Andronikov, Nikoleta Cimova, Marie Houskov

Zinc isotope systematics in snow and ice accretions in Central European mountains

Abstract not available.Petra Voldrichova, Vladislav Chrastny, Adela Sipkova, Juraj Farkas, Martin Novak, Marketa Stepanova, Michael Krachler, Frantisek Veselovsky, Vladimir Blaha, Eva Prechova, Arnost Komarek, Leona Bohdalkova, Jan Curik, Jitka Mikova, Lucie Erbanova, Petra Pacherov

Cu-Zn isotope constraints on the provenance of air pollution in Central Europe: using soluble and insoluble particles in snow and rime

Abstract not availableMartin Novak, Adela Sipkova, Vladislav Chrastny, Marketa Stepanova, Petra Voldrichova, Frantisek Veselovsky, Eva Prechova, Vladimir Blaha, Jan Curik, Juraj Farkas, Lucie Erbanova, Leona Bohdalkova, Jan Pasava, Jitka Mikova, Arnost Komarek, Michael Krachle

Common occurrence of a positive delta(53)Cr shift in Central European waters contaminated by geogenic/industrial chromium relative to source values

Carcinogenic effects of hexavalent chromium in waters are of concern in many countries worldwide. We explored Cr isotope systematics at 11 sites in the Czech Republic and Poland. Geogenic Cr pollution was associated with serpentinite bodies at former convergent plate margins, while anthropogenic Cr pollution resulted from electroplating, tanning, and the chemical industry. Cr(VI) concentration in geogenic waters was less than 40 ppb. Anthropogenic waters contained up to 127,000 ppb Cr(VI). At both geogenic and anthropogenic sites, where known, the source of pollution had a low δ⁵³Cr (<1‰). δ⁵³Cr of geogenic and anthropogenic waters was up to 3.9 and 5.8‰, respectively. At both serpentinite-dominated and industrial sites, δ⁵³Cr(VI)aq was shifted toward higher values, compared to the pollution source. At the industrial sites, this positive δ⁵³Cr shift was related to Cr(VI) reduction, a process known to fractionate Cr isotopes. At geogenic sites, the origin of high δ53Cr(VI)aq is tentatively ascribed to preferential release of ⁵³Cr during oxidation of soil Cr(III) and its mobilization to water. δ⁵³Cr(VI) of industrially contaminated waters was significantly higher (p<0.001) compared to δ⁵³Cr of waters carrying geogenic Cr(VI), implying that either the effective fractionation factor or process extent was greater for Cr(VI) reduction than for Cr(III) oxidation.Martin Novak, Vladislav Chrastny, Eva Cadkova, Juraj Farkas, Thomas D. Bullen, Jiri Tylcer, Zdenka Szurmanova, Marcel Cron, Eva Prechova, Jan Curik, Marketa Stepanova, Jan Pasava, Lucie Erbanova, Marie Houskova, Karel Puncochar, and Lucas A. Helleric

Calcium and strontium isotope dynamics in three polluted forest ecosystems of the Czech Republic, Central Europe

Calcium and strontium isotope ratios were used to identify Ca sources for bulk soil, soil solutions and runoff in the stressed forest ecosystems of Central Europe. All three study sites are underlain by Ca-poor crystalline bedrock (orthogneiss, leucogranite and serpentinite, respectively), but differ in historical rates of acid deposition. UDL, the spruce die-back affected site at the Czech–Polish border underlain by orthogneiss, received six times more acidifying compounds from the atmosphere than LYS and PLB, two paired catchments near the Czech–German border. The paired catchments near the Czech–German border, whose forests were only mildly damaged by industrial pollution, differ in their acid buffering capacity (extremely low for LYS leucogranite, extremely high for PLB serpentinite). At the spruce die-back affected catchment UDL, 19 years of monthly hydrochemical monitoring revealed six times higher atmospheric deposition and runoff fluxes of Ca, compared to the paired catchments LYS and PLB. Across the three sites, the mean δ⁴⁴Ca values increased in the order: spruce bark (−0.84‰) < spruce xylem (−0.31‰) < spruce fine roots (0.0‰) < bedrock (0.08‰) < soil (0.11‰) < spruce needles (0.31‰) < open-area precipitation (0.68‰) < spruce throughfall (0.71‰) < runoff (0.74‰) < soil water (1.11‰). At all three sites, Ca from atmospheric deposition was isotopically similar to Ca in runoff, indicating export of recent atmospheric Ca. All three catchments had isotopically lighter Ca in silicate bedrock and exported isotopically heavier Ca via runoff. Trees mostly represented the isotopically lightest Ca pool, whereas soil water collected by lysimeters contained isotopically heavy Ca. The ⁸⁷Sr/⁸⁶Sr isotope ratios were nearly uniform at the serpentinite site PLB (0.710). The ⁸⁷Sr/⁸⁶Sr ratios across the two felsic sites (UDL and LYS) increased in the order: open-area precipitation (0.710) < spruce canopy throughfall (0.715) < spruce xylem (0.723) < spruce needles (0.725) < soil water (0.734) < runoff (0.740) < soil (0.862) < bedrock (0.881). Collectively, the δ⁴⁴Ca and ⁸⁷Sr/⁸⁶Sr isotope systematics indicated that organic Ca cycling, along with atmospheric input of isotopically heavy Ca, largely contributed to the high-δ⁴⁴Ca values of runoff. The studied silicate bedrock had significantly lower δ⁴⁴Ca values than the reported world-wide average. While bulk soil Ca was likely affected by bedrock Ca even after the period of acid rain, the δ⁴⁴Ca difference between soil and soil water indicated a major role of recycled organic Ca in supplying nutrition to the trees. Future Ca-Sr isotope research in headwater catchments should include isotope analysis of sequentially leached Ca fractions from weathered minerals in soil to better assess the geogenic Ca inputs to runoff. In catchments currently recovering from pollution, Ca-Sr isotope fingerprinting of dust emitted from coal-fired power plants will be needed. Isotope analysis of local coal can indicate to what extent Ca-Sr isotope composition of past deposition contributes to the isotope signatures of present-day runoff.Martin Novak, Chris Holmden, Juraj Farkaš, Pavel Kram, Jakub Hruska, Jan Curik, Frantisek Veselovsky, Marketa Stepanova, Yulia V. Kochergina, Vojtech Erban, Daniela Fottova, Martin Simecek, Leona Bohdalkova, Eva Prechova, Petra Voldrichova, Vladimir Cernohou