Zinc Flux Research Articles

Intracellular zinc during cell activation and zinc deficiency

IntroductionZinc is an essential trace element having manifold functions within living cells. Zinc deficiency but also zinc excess impairs cell-specific functions whereas a balanced zinc level is required for an adequate cell behavior. Material and methodsThis study deals with the impact of cellular priming due to stimulation with interleukin (IL)-1, IL-2, IL-4, IL-6 or the chemokine CXCL12a and its subsequent influence on the intracellular free zinc concentration. Since cellular priming and activation is essential for proper immunological reactions, and across that highly cell-type specific, we investigated T cells, B cells, and peripheral blood mononuclear cells (PBMCs). Additionally, alterations of the intracellular zinc content was investigated by inducing zinc deficiency using the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethane-1,2-diamine (TPEN) with subsequent re-supplementation of zinc, hence generating an intracellular zinc flux. Evaluation of zinc staining with FluoZin3-AM, Zinpyr-1 and Zinquin was done by flow cytometry or by fluorescence microscopy. ResultsOur results indicate that cellular priming for different periods of time (10 minutes/one hour) causes decreased intracellular free zinc concentrations in the FluoZin3-AM staining and increased zinc concentrations stained with Zinpyr-1. Furthermore, zinc supplementation after induced zinc deficiency leads to a fast and excessive rise of the intracellular free zinc levels in most cellular compartments. ConclusionOur study emphasizes the importance of zinc homeostasis and zinc distribution during cellular priming and for certain signaling cascades especially in T and B cells. Moreover, we demonstrated that zinc re-supplementation of zinc deficient cells results in significantly elevated intracellular free zinc concentrations compared to untreated controls. Hence, this underlines the need of a balanced zinc homeostasis for proper immune cell function.

Metal ion fluxes controlling amphibian fertilization.

Mammalian oocytes undergo major changes in zinc content and localization in order to be fertilized, the most striking being the rapid exocytosis of over ten billion zinc ions, known as zinc sparks. Here we report that fertilization of amphibian Xenopus laevis eggs also initiates a zinc spark that progresses across the cell surface in coordination with dynamic calcium waves. This zinc exocytosis is accompanied by a newly recognized loss of intracellular manganese: synchrotron-based X-ray fluorescence and analytical electron microscopy reveal that zinc and manganese are sequestered in a system of cortical granules that are abundant at the animal pole. Through Electron-Nuclear Double-Resonance (ENDOR) studies, we rule out Mn2+ complexation with phosphate or nitrogenous ligands in intact eggs but the data are consistent with a carboxylate coordination environment. Our observations suggest that zinc and manganese fluxes are a conserved feature of fertilization in vertebrates and that they function as part of a physiological block to polyspermy.

Visualizing the Dynamic Metalation State of New Delhi Metallo-β-lactamase-1 in Bacteria Using a Reversible Fluorescent Probe.

New Delhi metallo-β-lactamase (NDM) grants resistance to a broad spectrum of β-lactam antibiotics, including last-resort carbapenems, and is emerging as a global antibiotic resistance threat. Limited zinc availability adversely impacts the ability of NDM-1 to provide resistance, but a number of clinical variants have emerged that are more resistant to zinc scarcity (e.g., NDM-15). To provide a novel tool to better study metal ion sequestration in host-pathogen interactions, we describe the development of a fluorescent probe that reports on the dynamic metalation state of NDM within Escherichia coli. The thiol-containing probe selectively coordinates the dizinc metal cluster of NDM and results in a 17-fold increase in fluorescence intensity. Reversible binding enables competition and time-dependent studies that reveal fluorescence changes used to detect enzyme localization, substrate and inhibitor engagement, and changes to metalation state through the imaging of live E. coli using confocal microscopy. NDM-1 is shown to be susceptible to demetalation by intracellular and extracellular metal chelators in a live-cell model of zinc dyshomeostasis, whereas the NDM-15 metalation state is shown to be more resistant to zinc flux. The development of this reversible turn-on fluorescent probe for the metalation state of NDM provides a new tool for monitoring the impact of metal ion sequestration by host defense mechanisms and for detecting inhibitor-target engagement during the development of therapeutics to counter this resistance determinant.

Sedimentary cycling and benthic fluxes of manganese, cobalt, nickel, copper, zinc and cadmium in the Peruvian oxygen minimum zone

Marine sediments are an important source and sink of bio-essential trace metals to the ocean. However, the different mechanisms leading to trace metal release or burial are not fully understood and the associated fluxes are not well quantified. Here, we present sediment, pore water, sequential extraction and benthic flux data of Mn, Co, Ni, Cu, Zn and Cd along a latitudinal depth transect across the Peruvian oxygen minimum zone at 12°S. Sediments are depleted in Mn and Co compared to the lithogenic background. Diffusive Mn fluxes from the sediments into the bottom water (−26 to −550 μmol m−2 y−1) are largely consistent with the rate of Mn loss from the solid phase (−100 to −1160 μmol m−2 yr−1) suggesting that 50% or more of the sedimentary Mn depletion is attributed to benthic efflux. In contrast, benthic Co fluxes (~ −3 μmol m−2 yr−1) are lower than the rate of Co loss from the solid phase (up to −120 μmol m−2 yr−1), implying Co dissolution in the water column. The trace metals Ni, Cu, Zn and Cd are enriched within the sediments with respect to the lithogenic background. Uptake of Ni by phytoplankton in the photic zone and delivery with organic matter to the sediment surface can account for up to 100% of the excess Ni accumulation (87 to 180 μmol m−2 y−1) in shelf sediments near the coast, whereas at greater water depth additional scavenging by Mn- and Fe-oxides may contribute to Ni accumulation. Up to 20% of excess Cu (33 to 590 μmol m−2 y−1) and generally less than 20% of excess Zn (58 to 2170 μmol m−2 y−1) and Cd (6 to 260 μmol m−2 y−1) can be explained by delivery with fresh organic matter. Sequential extraction data suggest that the discrepancies between the known sources of Cd (and Cu) and their excess accumulation may be driven by the delivery of allochthonous sulphide minerals precipitated from the water column. Additionally, Cu may be scavenged by downward sinking organic material. In contrast, precipitation of Zn sulphide chiefly takes place in the sediment. Diffusive Zn fluxes into the sediment (21 to 1990 μmol m−2 y−1) match the excess Zn accumulation suggesting that Zn delivery is mediated by molecular diffusion from bottom waters. Considering the diverse behavioural pattern of trace metals observed in this study, we argue that declining oxygen and increasing hydrogen sulphide concentrations in a future ocean will modify trace metal fluxes at the seafloor and the trace metal stoichiometry of seawater.

Overproduction of Human Zip (SLC39) Zinc Transporters in Saccharomyces cerevisiae for Biophysical Characterization.

Zinc constitutes the second most abundant transition metal in the human body, and it is implicated in numerous cellular processes, including cell division, DNA and protein synthesis as well as for the catalytic activity of many enzymes. Two major membrane protein families facilitate zinc homeostasis in the animal kingdom, i.e., Zrt/Irt-like proteins (ZIPs aka solute carrier 39, SLC39, family) and Zn transporters (ZnTs), essentially conducting zinc flux in the opposite directions. Human ZIPs (hZIPs) regulate import of extracellular zinc to the cytosol, being critical in preventing overaccumulation of this potentially toxic metal, and crucial for diverse physiological and pathological processes, including development of neurodegenerative disorders and several cancers. To date, our understanding of structure–function relationships governing hZIP-mediated zinc transport mechanism is scarce, mainly due to the notorious difficulty in overproduction of these proteins for biophysical characterization. Here we describe employment of a Saccharomyces cerevisiae-based platform for heterologous expression of hZIPs. We demonstrate that yeast is able to produce four full-length hZIP members belonging to three different subfamilies. One target (hZIP1) is purified in the high quantity and homogeneity required for the downstream biochemical analysis. Our work demonstrates the potential of the described production system for future structural and functional studies of hZIP transporters.

Zinc Dynamics during Drosophila Oocyte Maturation and Egg Activation.

SummaryTemporal fluctuations in zinc concentration are essential signals, including during oogenesis and early embryogenesis. In mammals, zinc accumulation and release are required for oocyte maturation and egg activation, respectively. Here, we demonstrate that zinc flux occurs in Drosophila oocytes and activated eggs, and that zinc is required for female fertility. Our synchrotron-based X-ray fluorescence microscopy reveals zinc as the most abundant transition metal in Drosophila oocytes. Its levels increase during oocyte maturation, accompanied by the appearance of zinc-enriched intracellular granules in the oocyte, which depend on transporters. Subsequently, in egg activation, which mediates the transition from oocyte to embryo, oocyte zinc levels decrease significantly, as does the number of zinc-enriched granules. This pattern of zinc dynamics in Drosophila oocytes follows a similar trajectory to that in mammals, extending the parallels in female gamete processes between Drosophila and mammals and establishing Drosophila as a model for dissecting reproductive roles of zinc.

SUN-736 Knockout of Membrane Androgen Receptor ZIP9 Results in Reduced Female Fecundity and Abnormal Egg Activation in Zebrafish

Recently, our research group cloned and characterized a putative membrane androgen receptor from teleost ovarian tissue that was found to be homologous with the zinc transporter protein ZIP9 (Slc39a9). To date, ZIP9 is the only zinc transporter that is known to be ligand activated or possess steroid receptor activity. Since the discovery of its androgen receptor activity, ZIP9 has been found to mediate androgen actions in a variety of tissues including teleost ovarian follicle cells, human cancer cell lines, and murine Sertoli cells. However, ZIP9 has not been examined in an in vivo model so the precise physiological functions of this receptor remain unclear. A ZIP9-mutant strain of zebrafish was developed using a CRISPR-Cas9 system in order to examine the role of the protein in teleost reproduction. While ZIP9-mutant males had similar breeding occurrence and fertilization rates to wild-type fish, mutant females exhibited severe reductions in fecundity compared to wild-type fish. ZIP9-mutant females spawn significantly fewer eggs of which a high proportion failed to undergo chorion elevation, a characteristic of normal egg activation. Eggs that showed this failed chorion elevation phenotype had significantly lower fertilization rates and produced larvae that exhibit a high incidence of pericardial/yolk sac edema and reduced growth compared to larvae hatched from wild-type eggs. However, no differences were observed in the proportions of oocytes at later stages of development between ZIP9-mutant and wild-type fish, suggesting the observed phenotypes are not related to abnormal oogenesis. We observed that mature wild-type eggs have numerous cortically located vesicles that are autofluorescent under ultraviolet light and decrease in number when the eggs undergo activation, suggesting they undergo exocytosis during the cortical reaction. While zinc is known to be stored in vesicles that undergo exocytosis in mammalian eggs, the role of zinc in teleost egg activation is currently unknown. In eggs from wild-type fish, we observed an increase in extracellular zinc levels upon egg activation and treatment with a zinc ionophore (zinc pyrithione) significantly reduced the number of eggs that undergo normal chorion elevation when activated. This suggests a role for zinc in zebrafish egg activation similar to that observed in mammals. Of interest, ZIP9-mutant eggs that did not undergo chorion elevation had significantly smaller vesicles than those found in wild-type fish eggs. This abnormal vesicle morphology and failure to undergo chorion elevation suggest a role of ZIP9 in egg activation. Additional insight into the role of zinc in zebrafish egg activation and the mechanism by which ZIP9 disruption leads to abnormal cortical vesicles and egg activation will help determine if ZIP9 plays a role in zinc transport and flux in zebrafish eggs during activation.

TMEM163 (ZNT11) protein interacts with distinct ZNT efflux transporters

Our recent work showed that TMEM163 is a zinc efflux transporter that belongs to the cation diffusion facilitator (CDF) family of proteins. We propose that the TMEM163 be now called ZNT11 as a new member of the Group 1 CDF family ZNT efflux proteins that include 10 mammalian zinc transporters, ZNT1‐ZNT10. Accordingly, we hypothesize that TMEM163/ZNT11 interacts with at least one of the ZNT proteins, namely ZNT1 and ZNT2, based on its structural and functional characteristics. To show that TMEM163/ZNT11 interacts with either ZNT1 or ZNT2, we cloned their cDNAs into mammalian expression constructs containing either Myc‐DDK or HA peptide tag. Using HEK‐293 cells, we individually transfected TMEM163/ZNT11, ZNT1, and ZNT2 constructs as negative controls, while we co‐transfected TMEM163/ZNT11 and ZNT1, or TMEM163/ZNT11 and ZNT2. As a positive control, we co‐transfected HA‐tagged TMEM163/ZNT11 with a Myc‐DDK‐tagged TMEM163/ZNT11 construct in HEK‐293 cells, based on a previous report that its rodent counterpart exists as a homodimer. We then used a co‐immunoprecipitation assay using either anti‐HA‐ or anti‐DDK‐bound agarose beads to pull down one of the target proteins. Western blot analysis showed that TMEM163/ZNT11 physically bound ZNT1 or ZNT2 protein. To determine the functional relevance of the interaction, we performed zinc flux assays using two zinc‐specific fluorescence dyes, Fluozin‐3 (high affinity, membrane impermeable) and Newport Green (low affinity, membrane permeable) following single‐ and co‐transfection of TMEM163/ZNT11, ZNT1, and ZNT2 constructs in HeLa cells. Our results confirmed that homodimers of TMEM163/ZNT11, ZNT1, and ZNT2 proteins transport zinc out of the cells, but that the efflux activity of homodimer TMEM163/ZNT11 proteins varied slightly in magnitude when compared with heterodimers of TMEM163/ZNT11 and ZNT1, or TMEM163/ZNT11 and ZNT2 proteins. These results suggest that the interaction between TMEM163/ZNT11 and distinct ZNT proteins is physiologically relevant and may serve to modify the transport activity of ZNT protein interactor. Overall, our investigations showed for the first time that TMEM163/ZNT11 forms functional heterodimers with ZNT1 and ZNT2 proteins. Thus, TMEM163/ZNT11 by itself, or in combination with one of these specific ZNT proteins, may play a crucial role in maintaining intracellular zinc homeostasis in specific cell types.Support or Funding InformationThis work is funded by NIH R15 NS101594.

One-Step Synthesis of Graphene, Copper and Zinc Oxide Graphene Hybrids via Arc Discharge: Experiments and Modeling

In this paper, we report on a modified arc process to synthetize graphene, copper and zinc oxide graphene hybrids. The anode was made of pure graphite or graphite mixed with metals or metal oxides. After applying a controlled direct current, plasma is created in the interelectrode region and the anode is consumed by eroding. Continuous and abundant flux of small carbon, zinc or copper species, issued from the anode at a relatively high temperature, flows through the plasma and condenses in the vicinity of a water-cooled cathode leading to few-layered graphene sheets and highly ordered carbon structures. When the graphite rod is filled with copper or zinc oxide nanoparticles, few layers of curved graphene films were anchored with spherical Cu and ZnO nanoparticles leading to a one-step process synthesis of graphene hybrids, which combine the synergetic properties of graphene along with nanostructured metals or semiconducting materials. The as-prepared samples were characterized by Raman spectroscopy, X-ray diffraction (XRD), spatially resolved electron energy loss spectroscopy (EELS), energy filtered elemental mapping and transmission electron microscopy (TEM). In addition to the experimental study, numerical simulations were performed to determine the velocity, temperature and chemical species distributions in the arc plasma under specific graphene synthesis conditions, thereby providing valuable insight into growth mechanisms.

Sperm Cohort-Specific Zinc Signature Acquisition and Capacitation-Induced Zinc Flux Regulate Sperm-Oviduct and Sperm-Zona Pellucida Interactions.

Building on our recent discovery of the zinc signature phenomenon present in boar, bull, and human spermatozoa, we have further characterized the role of zinc ions in the spermatozoa’s pathway to fertilization. In boar, the zinc signature differed between the three major boar ejaculate fractions, the initial pre-rich, the sperm-rich, and the post-sperm-rich fraction. These differences set in the sperm ejaculatory sequence establish two major sperm cohorts with marked differences in their sperm capacitation progress. On the subcellular level, we show that the capacitation-induced Zn-ion efflux allows for sperm release from oviductal glycans as analyzed with the oviductal epithelium mimicking glycan binding assay. Sperm zinc efflux also activates zinc-containing enzymes and proteases involved in sperm penetration of the zona pellucida, such as the inner acrosomal membrane matrix metalloproteinase 2 (MMP2). Both MMP2 and the 26S proteasome showed severely reduced activity in the presence of zinc ions, through studies using by gel zymography and the fluorogenic substrates, respectively. In the context of the fertilization-induced oocyte zinc spark and the ensuing oocyte-issued polyspermy-blocking zinc shield, the inhibitory effect of zinc on sperm-borne enzymes may contribute to the fast block of polyspermy. Altogether, our findings establish a new paradigm on the role of zinc ions in sperm function and pave the way for the optimization of animal semen analysis, artificial insemination (AI), and human male-factor infertility diagnostics.

Control of polymorphism during epitaxial growth of hyperferroelectric candidate LiZnSb on GaSb (111)B

A major challenge for ferroelectric devices is the depolarization field, which competes with and often destroys long-range polar order in the limit of ultrathin films. Recent theoretical predictions suggest a new class of materials, termed hyperferroelectics, that should be robust against the depolarization field and enable ferroelectricity down to the monolayer limit. Here, the authors demonstrate the epitaxial growth of hexagonal LiZnSb, one of the hyperferroelectric candidate materials, by molecular-beam epitaxy on GaSb (111)B substrates. Due to the high volatility of all three atomic species, they find that LiZnSb can be grown in an adsorption-controlled window, using an excess zinc flux. Within this window, the desired polar hexagonal phase is stabilized with respect to a competing cubic polymorph, as revealed by x-ray diffraction and transmission electron microscopy measurements. First-principles calculations show that for moderate amounts of epitaxial strain and moderate concentrations of Li vacancies, the cubic LiZnSb phase is lower in formation energy than the hexagonal phase, but only by a few millielectronvolts per formula unit. Therefore, they suggest that kinetics plays a role in stabilizing the desired hexagonal phase at low temperatures. Their results provide a path toward experimentally demonstrating ferroelectricity and hyperferroelectricity in a new class of ternary intermetallic compounds.

Zinc supplementation improves the harvest purity of β-glucuronidase from CHO cell culture by suppressing apoptosis.

The variability of trace metals in cell culture media is a potential manufacturing concern because it may significantly affect the production and quality of therapeutic proteins. Variability in trace metals in CHO cell culture has been shown to impact critical production metrics such as cell growth, viability, nutrient consumption, and production of recombinant proteins. To better understand the influence of excess supplementation, zinc and copper were initially supplemented with 50-μM concentrations to determine the impact on the production and quality of β-glucuronidase, a lysosomal enzyme, in a parallel bioreactor system. Ethylenediaminetetraacetic acid (EDTA), a metal chelator, was included as another treatment to induce a depletion of trace metal bioavailability to examine deficiency. Samples were drawn daily to monitor cell growth and viability, nutrient levels, β-glucuronidase activity, and trace zinc flux. Cell cycle analysis revealed the inhibition of sub-G0/G1 species in zinc supplemented cultures, maintaining higher viability compared to the control, EDTA-, and copper-supplemented cultures. Enzyme activity analysis in the harvests revealed higher specific activity of β-glucuronidase in reactors supplemented with zinc. A confirmation run was conducted with supplementations of zinc at concentrations of 50, 100, and 150μM. Further cell cycle analysis and caspase-3 analysis demonstrated the role of zinc as an apoptosis suppressor responsible for the enhanced harvest purity of β-glucuronidase from zinc-supplemented bioreactors.

Laboratory and Field-Based Assessment of the Effects of Sediment Capping Materials on Zinc Flux, Bioavailability, and Toxicity.

A former mining site has been the subject of extensive remediation and restoration, with a significant focus on disconnecting mine spoils from groundwater and managing the quantity and quality of runoff. A remaining task is ensuring concentrations of zinc (Zn) in the stream outflow of a pit lake are reduced below water quality standards. The efficacy of multiple capping materials for decreasing Zn dissolution from sediments was conducted under natural and reasonable worst-case conditions (pH = 5.5). Capping materials included AquaBlok™, limestone, and limestone-bone char. Field exposures were conducted in limnocorrals that isolated overlying water columns above the sediment and capping treatments. Simultaneous in situ and ex situ toxicity tests were conducted using Daphnia magna, Hyalella azteca, and Chironomus dilutus. In situ caged organisms were protected from temperature shock (warm epilimnetic waters) by deploying within a Toxicity Assessment Container System (TACS). Organisms were exposed to surficial sediments, caps, and hypolimnetic overlying waters for 4 d. Ex situ testing was conducted in core tube mesocosms containing sediments and caps at similar temperatures (15-19 °C). Results demonstrated the usefulness of TACS deployment in stratified lake systems. There were no differences in responses between treatments involving sediment capping materials in both in situ and ex situ tests. The lack of differences was likely due to dissolved Zn in surface water being below the hardness-adjusted threshold effects levels (164 μg L-1 ). This field- and laboratory-based weight-of-evidence study provided site-specific data to support the selection of an effective remedy, with reduced uncertainty compared to laboratory and chemistry-only approaches. Environ Toxicol Chem 2019;39:240-249. © 2019 SETAC.

Dissolved organic matter and trace element variability in a blackwater-fed bay following precipitation

Dissolved organic matter (DOM) often forms complexes with trace metals. The main objective of this study was to identify the sources of trace elements to a coastal bay that is fed by two blackwater rivers using DOM compositions. Surface water samples from twelve sites in Weeks Bay, Alabama were collected during four field trips and a bottom sediment sample was collected during one of the trips. Spectroscopic measurements in tandem with parallel factor analysis and multivariate statistics were used to derive DOM compositions and inductively coupled plasma mass spectrometry was used for determining trace metal concentrations. DOM chemistry and trace element concentrations together with precipitation and discharge, watershed land use and land cover data, and physicochemical parameters were used to determine the source of trace elements in the adjoining areas of the watershed to the bay and finally settling into the bay sediments. Arsenic, copper, and uranium fluxes increased while fluxes of mercury, zinc, manganese, and iron decreased following precipitation events. Concentration of all trace elements analyzed were 10,000 times higer in the bay botton sediments than their concentrations in the water colunmn. Microbially reprocessed and soil derived DOM components increased along with the trace elements and nutrients in the bay as a result of precipitation whereas concentration of terrestrial humic-like DOM component remained the same during both dry (spring) and wet (summer and fall) periods. Principal component analysis revealed significant correlations between microbially reprocessed DOM and the trace elements including arsenic, copper and uranium indicating urban, pasture, and agricultural areas in the watershed were the sources for these trace metals. On the other hand, multiple sources were identified for manganese, zinc, and iron. Overall it was found that DOM compositions are useful for inferring the possible sources and mobility of trace elements in aquatic systems.

Interrogating Intracellular Zinc Chemistry with a Long Stokes Shift Zinc Probe ZincBY-4.

Previous work has shown that fluctuations in zinc content and subcellular localization play key roles in regulating cell cycle progression; however, a deep mechanistic understanding requires the determination of when, where, and how labile zinc pools are concentrated into or released from stores. Labile zinc ions can be difficult to detect with probes that require hydrolysis of toxic protecting groups or application at high concentrations that negatively impact cell function. We previously reported a BODIPY-based zinc probe, ZincBY-1, that can be used at working concentrations that are 20-200-fold lower than concentrations employed with other probes. To better understand how zinc pools can be visualized at such low probe concentrations, we modulated the photophysical properties via changes at the 5-position of the BODIPY core. One of these, ZincBY-4, exhibits an order of magnitude higher affinity for zinc, an 8-fold increase in brightness in response to zinc, and a 100 nm Stokes shift within cells. The larger Stokes shift of ZincBY-4 presents a unique opportunity for simultaneous imaging with GFP or fluorescein sensors upon single excitation. Finally, by creating a proxy for the cellular environment in spectrometer experiments, we show that the ZincBY series are highly effective at 50 nM because they can pass membranes and accumulate in regions of high zinc concentration within a cell. These features of the ZincBY probe class have widespread applications in imaging and for understanding the regulatory roles of zinc fluxes in live cells.

The Zinc Transporter Zip7 Is Downregulated in Skeletal Muscle of Insulin-Resistant Cells and in Mice Fed a High-Fat Diet.

Background: The zinc transporter Zip7 modulates zinc flux and controls cell signaling molecules associated with glucose metabolism in skeletal muscle. The present study evaluated the role of Zip7 in cell signaling pathways involved in insulin-resistant skeletal muscle and mice fed a high-fat diet. Methods: Insulin-resistant skeletal muscle cells were prepared by treatment with an inhibitor of the insulin receptor, HNMPA-(AM)3 or palmitate, and Zip7 was analyzed along with pAkt, pTyrosine and Glut4. Similarly, mice fed normal chow (NC) or a high-fat diet (HFD) were also analyzed for protein expression of Glut4 and Zip7. An overexpression system for Zip7 was utilized to determine the action of this zinc transporter on several genes implicated in insulin signaling and glucose control. Results: We identified that Zip7 is upregulated by glucose in normal skeletal muscle cells and downregulated in insulin-resistant skeletal muscle. We also observed (as expected) a decrease in pAkt and Glut4 in the insulin-resistant skeletal muscle cells. The overexpression of Zip7 in skeletal muscle cells led to the modulation of key genes involved in the insulin signaling axis and glucose metabolism including Akt3, Dok2, Fos, Hras, Kras, Nos2, Pck2, and Pparg. In an in vivo mouse model, we identified a reduction in Glut4 and Zip7 in the skeletal muscle of mice fed a HFD compared to NC controls. Conclusions: These data suggest that Zip7 plays a role in skeletal muscle insulin signaling and is downregulated in an insulin-resistant, and HFD state. Understanding the molecular mechanisms of Zip7 action will provide novel opportunities to target this transporter therapeutically for the treatment of insulin resistance and type 2 diabetes.

Bovine eggs release zinc in response to parthenogenetic and sperm-induced egg activation

Upon fertilization or parthenogenesis, zinc is released into the extracellular space through a series of exocytic events termed zinc sparks, which are tightly coordinated with intracellular calcium transients. The zinc spark reduces the total amount of intracellular zinc, and this reduction is necessary and sufficient to induce egg activation even in the absence of calcium transients. In addition, this zinc release contributes to the block to polyspermy through modification of the zona pellucida. The zinc spark has been documented in all organisms examined to date including the mouse, two species of nonhuman primates, and human. Here we determined whether zinc sparks occur in the bovine, an important model of gamete development in mono-ovulatory mammalian species. We obtained metaphase II-arrested (MII) bovine eggs following in vitro maturation. Total zinc, assessed in single cells using X-Ray Fluorescence Microscopy, was significantly more abundant in the bovine egg compared to iron and copper. Studies with intracellular fluorescent probes revealed that labile zinc pools are localized to discrete cytoplasmic punctae enriched at the cortex. To determine whether zinc undergoes dynamic fluxes during egg activation, we parthenogenetically activated bovine eggs using two approaches: ionomycin or bovine phospholipase C zeta (bPlcζ). Both these methods induced zinc sparks coordinately with intracellular calcium transients. The zinc spark was also observed in bovine eggs following intracytoplasmic sperm injection. These results establish that zinc is the most abundant transition metal in the bovine egg, and zinc flux during egg activation - induced by chemical activation or sperm - is a highly conserved event across mammalian species.

Diffusive fluxes of phosphorus, potassium and metallic microelements as affected by soil compaction

ABSTRACTThe objective was to evaluate the effects of soil compaction on phosphorus (P), potassium (K), zinc (Zn), copper (Cu), iron (Fe) and manganese (Mn) diffusive fluxes. The experiment consisted of two Oxisols and eight compaction pressures. The soil samples were placed in diffusion chambers, simultaneously with two ion-exchange membranes (anionic and cationic), compressed and incubated for 20 days. The P, K, Zn, Cu, Fe, and Mn diffusive fluxes (PDF, KDF, ZnDF, CuDF, FeDF, and MnDF) were determined. The compaction decreased the PDF in the oxidic-gibbsitic soil, and increased KDF, ZnDF, CuDF, and MnDF, in both soils. There was a higher diffusion of Zn, Cu, and Mn in the kaolinitic than the oxidic-gibbsitic soil. The descending order of cationic-microelement diffusive flux was MnDF > ZnDF ⋙ FeDF > CuDF. Presumably, Fe was mainly diffused as organic complexes with net negative charges, whereas Zn and Mn as free ions and, or, inorganic and organic complexes with positive charges.Abbreviations: CuDF, cupper diffusive flux; FeDF, iron diffusive flux; KDF, potassium diffusive flux; MnDF, manganese diffusive flux; PDF, phosphorus diffusive flux; ZnDF, zinc diffusive flux; AEM, anion-exchange membrane; CEM, cation-exchange membrane

Cyclic AMP Pathway Activation and Extracellular Zinc Induce Rapid Intracellular Zinc Mobilization in Candida albicans.

Zinc is an essential micronutrient, required for a range of zinc-dependent enzymes and transcription factors. In mammalian cells, zinc serves as a second messenger molecule. However, a role for zinc in signaling has not yet been established in the fungal kingdom. Here, we used the intracellular zinc reporter, zinbo-5, which allowed visualization of zinc in the endoplasmic reticulum and other components of the internal membrane system in Candida albicans. We provide evidence for a link between cyclic AMP/PKA- and zinc-signaling in this major human fungal pathogen. Glucose stimulation, which triggers a cyclic AMP spike in this fungus resulted in rapid intracellular zinc mobilization and this “zinc flux” could be stimulated with phosphodiesterase inhibitors and blocked via inhibition of adenylate cyclase or PKA. A similar mobilization of intracellular zinc was generated by stimulation of cells with extracellular zinc and this effect could be reversed with the chelator EDTA. However, zinc-induced zinc flux was found to be cyclic AMP independent. In summary, we show that activation of the cyclic AMP/PKA pathway triggers intracellular zinc mobilization in a fungus. To our knowledge, this is the first described link between cyclic AMP signaling and zinc homeostasis in a human fungal pathogen.

Zinc transporters belonging to the Cation Diffusion Facilitator (CDF) family have complementary roles in transporting zinc out of the cytosol

Zinc is an essential trace element that is required for the function of a large number of proteins. As these zinc-binding proteins are found within the cytosol and organelles, all eukaryotes require mechanisms to ensure that zinc is delivered to organelles, even under conditions of zinc deficiency. Although many zinc transporters belonging to the Cation Diffusion Facilitator (CDF) families have well characterized roles in transporting zinc into the lumens of intracellular compartments, relatively little is known about the mechanisms that maintain organelle zinc homeostasis. The fission yeast Schizosaccharomyces pombe is a useful model system to study organelle zinc homeostasis as it expresses three CDF family members that transport zinc out of the cytosol into intracellular compartments: Zhf1, Cis4, and Zrg17. Zhf1 transports zinc into the endoplasmic reticulum, and Cis4 and Zrg17 form a heterodimeric complex that transports zinc into the cis-Golgi. Here we have used the high and low affinity ZapCY zinc-responsive FRET sensors to examine cytosolic zinc levels in yeast mutants that lack each of these CDF proteins. We find that deletion of cis4 or zrg17 leads to higher levels of zinc accumulating in the cytosol under conditions of zinc deficiency, whereas deletion of zhf1 results in zinc accumulating in the cytosol when zinc is not limiting. We also show that the expression of cis4, zrg17, and zhf1 is independent of cellular zinc status. Taken together our results suggest that the Cis4/Zrg17 complex is necessary for zinc transport out of the cytosol under conditions of zinc-deficiency, while Zhf1 plays the dominant role in removing zinc from the cytosol when labile zinc is present. We propose that the properties and/or activities of individual CDF family members are fine-tuned to enable cells to control the flux of zinc out of the cytosol over a broad range of environmental zinc stress.