47,549 research outputs found

    Polar localization of plasma membrane Ca2+/Mg2+ ATPase correlates with the pattern of steady ionic currents in eggs of Lymnaea stagnalis and Bithynia tentaculata (Mollusca)

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    During extrusion of the first polar body in eggs of Lymnaea stagnalis and Bithynia tentaculata a localized Ca2+ /Mg2+ ATPase activity was detected, using Ando's enzyme-cytochemical method for electron microscopy [Ando et al. (1981) Acta Histochem Cytochem 14:705-726]. The enzyme activity was distributed in a polar fashion, along the cytoplasmic face of the plasma membrane. In the eggs of Lymnaea it was found only in the vegetal hemisphere, whereas in Bithynia eggs it was localized both in the vegetal hemisphere and at the animal pole. This pattern of enzyme activity corresponds to the polar pattern of transcellular ionic currents measured with the vibrating probe, which we showed to be partially carried or regulated by calcium [Zivkovic and Dohmen (1989) Biol Bull (Woods Hole) 176 (Suppl):103-109]. The characteristics of the ATPase were studied using a variety of approaches such as ion and substrate depletions and substitutions, addition of specific inhibitors of ATPase activity, treatment with EDTA/EGTA and electron energy-loss spectrometry. The results indicate that, in Lymnaea, there are at least two enzymatic entities. The first one is a Ca2+ /Mg2+ ATPase localized along the membrane and in the cortex of the vegetal hemisphere. The second one is a Ca2+-stimulated ATPase (calcium pump of the plasma membrane) localized in a small region of the membrane at the vegetal pole. We speculate that in the eggs of Lymnaea and Bithynia a functional relationship exists between the plasma-membrane-associated ATPase activity and the transcellular ionic currents measured in the same region

    Exploring emergent properties in cellular homeostasis using OnGuard to model K+ and other ion transport in guard cells

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    It is widely recognized that the nature and characteristics of transport across eukaryotic membranes are so complex as to defy intuitive understanding. In these circumstances, quantitative mathematical modeling is an essential tool, both to integrate detailed knowledge of individual transporters and to extract the properties emergent from their interactions. As the first, fully integrated and quantitative modeling environment for the study of ion transport dynamics in a plant cell, OnGuard offers a unique tool for exploring homeostatic properties emerging from the interactions of ion transport, both at the plasma membrane and tonoplast in the guard cell. OnGuard has already yielded detail sufficient to guide phenotypic and mutational studies, and it represents a key step toward ‘reverse engineering’ of stomatal guard cell physiology, based on rational design and testing in simulation, to improve water use efficiency and carbon assimilation. Its construction from the HoTSig libraries enables translation of the software to other cell types, including growing root hairs and pollen. The problems inherent to transport are nonetheless challenging, and are compounded for those unfamiliar with conceptual ‘mindset’ of the modeler. Here we set out guidelines for the use of OnGuard and outline a standardized approach that will enable users to advance quickly to its application both in the classroom and laboratory. We also highlight the uncanny and emergent property of OnGuard models to reproduce the ‘communication’ evident between the plasma membrane and tonoplast of the guard cell

    Plasma membrane calcium ATPase activity is regulated by actin oligomers through direct interaction

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    As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca2+ with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca2+-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca2+-ATPase activity was related to an increase in the apparent affinity for Ca2+ and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca2+ homeostasis.Fil: Dalghi, Marianela Gisela. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de QuĂ­mica y FĂ­sico-QuĂ­mica BiolĂłgicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica. Instituto de QuĂ­mica y FĂ­sico-QuĂ­mica BiolĂłgicas; ArgentinaFil: FernĂĄndez, Marisa Mariel. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Ferreira Gomes, Mariela Soledad. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de QuĂ­mica y FĂ­sico-QuĂ­mica BiolĂłgicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica. Instituto de QuĂ­mica y FĂ­sico-QuĂ­mica BiolĂłgicas; ArgentinaFil: Mangialavori, Irene Cecilia. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de QuĂ­mica y FĂ­sico-QuĂ­mica BiolĂłgicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica. Instituto de QuĂ­mica y FĂ­sico-QuĂ­mica BiolĂłgicas; ArgentinaFil: Malchiodi, Emilio Luis. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de QuĂ­mica y FĂ­sico-QuĂ­mica BiolĂłgicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica. Instituto de QuĂ­mica y FĂ­sico-QuĂ­mica BiolĂłgicas; ArgentinaFil: Strehler, Emanuel E.. Mayo Clinic College of Medicine; Estados UnidosFil: Rossi, Juan Pablo Francisco. Consejo Nacional de Investigaciones CientĂ­ficas y TĂ©cnicas. Oficina de CoordinaciĂłn Administrativa Houssay. Instituto de QuĂ­mica y FĂ­sico-QuĂ­mica BiolĂłgicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y BioquĂ­mica. Instituto de QuĂ­mica y FĂ­sico-QuĂ­mica BiolĂłgicas; Argentin

    Quantitative description of ion transport via plasma membrane of yeast and small cells

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    Modeling of ion transport via plasma membrane needs identification and quantitative understanding of the involved processes. Brief characterization of main ion transport systems of a yeast cell (Pma1, Ena1, TOK1, Nha1, Trk1, Trk2, non-selective cation conductance) and determining the exact number of molecules of each transporter per a typical cell allow us to predict the corresponding ion flows. In this review a comparison of ion transport in small yeast cell and several animal cell types is provided. The importance of cell volume to surface ratio is emphasized. The role of cell wall and lipid rafts is discussed in respect to required increase in spatial and temporal resolution of measurements. Conclusions are formulated to describe specific features of ion transport in a yeast cell. Potential directions of future research are outlined based on the assumptions.Comment: 22 pages, 6 figures, 1 tabl

    The mechanisms of calcium homeostasis and signalling in the lens

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    Excessive Ca2+ can be detrimental to cells and raised levels of Ca2+ in human lenses with cortical cataract have been found to play a major role in the opacification process. Ca2+ homeostasis is therefore, recognised as having fundamental importance in lens pathophysiology. Furthermore, Ca2+ plays a central role as a second messenger in cell signalling and mechanisms have evolved which give cells exquisite control over intracellular Ca2+ ([Ca2+]i) via an array of specialised regulatory and signalling proteins. In this review we discuss these mechanisms as they apply to the lens. Ca2+ levels in human aqueous humour are approximately 1 mM and there is a large, 10,000 fold, inwardly directed gradient across the plasma membrane. In the face of such a large gradient highly efficient mechanisms are needed to maintain low [Ca2+]i. The Na+/Ca2+ exchanger (NCX) and plasma membrane Ca2+-ATPase (PMCA) actively remove Ca2+ from the cells, whereas the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) sequesters Ca2+ in the endoplasmic reticulum (ER) Ca2+ store. In lens epithelial cells the dominant role is played by the ATPases, whilst in the fibre cells NCX activity appears to be more important. Usually, [Ca2+]i can be increased in a number of ways. Ca2+ influx through the plasma membrane, for example, is mediated by an array of channels with evidence in the lens for the presence of voltage-operated Ca2+ channels (VOCCs), receptor-operated Ca2+ channels (ROCCs) and channels mediating store-operated Ca2+ entry (SOCE). Ca2+ signalling is initiated via activation of G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTK) of which the lens expresses a surprisingly diverse array responding to various neurotransmitters, hormones, growth factors, autocoids and proteases. Downstream of plasma membrane receptors are IP3-gated channels (IP3Rs) and ryanodine receptors (RYRs) located in the ER, which when activated cause a rapid increase in [Ca2+]i and these have also been identified in the lens. Through an appreciation of the diversity and complexity of the mechanisms involved in Ca2+ homeostasis in normal lens cells we move closer to an understanding of the mechanisms which mediate pathological Ca2+ overload as occurs in the process of cataract formation

    Systems analysis of guard cell membrane transport for enhanced stomatal dynamics and water use efficiency

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    Stomatal transpiration is at the centre of a crisis in water availability and crop production that is expected to unfold over the next 20-30 years. Global water usage has increased 6-fold in the past 100 years, twice as fast as the human population, and is expected to double again before 2030, driven mainly by irrigation and agriculture. Guard cell membrane transport is integral to controlling stomatal aperture and offers important targets for genetic manipulation to improve crop performance. However, its complexity presents a formidable barrier to exploring such possibilities. With few exceptions, mutations that increase water use efficiency commonly have been found to do so with substantial costs to the rate of carbon assimilation, reflecting the trade-off in CO2 availability with suppressed stomatal transpiration. One approach yet to be explored in any detail relies on quantitative systems analysis of the guard cell. Our deep knowledge of transport and homeostasis in these cells gives real substance to the prospect for ‘reverse engineering’ of stomatal responses, using in silico design in directing genetic manipulation for improved water use and crop yields. Here we address this problem with a focus on stomatal kinetics, taking advantage of the OnGuard software and models of the stomatal guard cell (www.psrg.org.uk) recently developed for exploring stomatal physiology. Our analysis suggests that manipulations of single transporter populations are likely to have unforeseen consequences. Channel gating, especially of the dominant K+ channels, appears the most favorable target for experimental manipulation

    A proteomic and phosphoproteomic analysis of Oryza sativa plasma membrane and vacuolar membrane

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    Proteomic and phosphoproteomic analyses of rice shoot and root tonoplast-enriched and plasma membrane-enriched membrane fractions were carried out to look at tissue-specific expression, and to identify putative regulatory sites of membrane transport proteins. Around 90 unique membrane proteins were identified, which included primary and secondary transporters, ion channels and aquaporins. Primary H+ pumps from the AHA family showed little isoform specificity in their tissue expression pattern, whereas specific isoforms of the Ca2+ pump ECA/ACA family were expressed in root and shoot tissues. Several ABC transporters were detected, particularly from the MDR and PDR subfamilies, which often showed expression in either roots or shoots. Ammonium transporters were expressed in root, but not shoot, tissue. Large numbers of sugar transporters were expressed, particularly in green tissue. The occurrence of phosphorylation sites in rice transporters such as AMT1;1 and PIP2;6 agrees with those previously described in other species, pointing to conserved regulatory mechanisms. New phosphosites were found in many transporters, including H+ pumps and H+:cation antiporters, often at residues that are well conserved across gene families. Comparison of root and shoot tissue showed that phosphorylation of AMT1;1 and several further transporters may be tissue dependent

    Processing of mitochondrial precursor proteins

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