ADP-Ribosylation Factor 1 Transiently Activates High-Affinity Adaptor Protein Complex AP-1 Binding Sites On Golgi Membranes

Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Substituting GTP for GTPγS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 μM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1·GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37°C, whereas AP-1 recruited with GTPγS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlF_n, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1·GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1·GTP first primes the Golgi membrane at 37°C, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1·GTP hydrolysis point. Thus, hydrolysis of ARF1·GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.     

Intracellular Cl? Dependence of Na-H Exchange in Barnacle Muscle Fibers under Normotonic and Hypertonic Conditions

We previously showed that shrinking a barnacle muscle fiber (BMF) in a hypertonic solution (1,600 mosM/kg) stimulates an amiloride-sensitive Na-H exchanger. This activation is mediated by a G protein and requires intracellular Cl⁻. The purpose of the present study was to determine (a) whether Cl⁻ plays a role in the activation of Na-H exchange under normotonic conditions (975 mosM/kg), (b) the dose dependence of [Cl⁻]_i for activation of the exchanger under both normo- and hypertonic conditions, and (c) the relative order of the Cl⁻- and G-protein-dependent steps. We acid loaded BMFs by internally dialyzing them with a pH-6.5 dialysis fluid containing no Na⁺ and 0–194 mM Cl⁻. The artificial seawater bathing the BMF initially contained no Na⁺. After dialysis was halted, adding 50 mM Na⁺ to the artificial seawater caused an amiloride-sensitive pH_i increase under both normo- and hypertonic conditions. The computed Na-H exchange flux (J _Na-H) increased with increasing [Cl⁻]_i under both normo- and hypertonic conditions, with similar apparent K _m values (∼120 mM). However, the maximal J _Na-H increased by nearly 90% under hypertonic conditions. Thus, activation of Na-H exchange at low pH_i requires Cl⁻ under both normo- and hypertonic conditions, but at any given [Cl⁻]_i, J _Na-H is greater under hyper- than normotonic conditions. We conclude that an increase in [Cl⁻]_i is not the primary shrinkage signal, but may act as an auxiliary shrinkage signal. To determine whether the Cl⁻-dependent step is after the G-protein-dependent step, we predialyzed BMFs to a Cl⁻-free state, and then attempted to stimulate Na-H exchange by activating a G protein. We found that, even in the absence of Cl⁻, dialyzing with GTPγS or AlF₃, or injecting cholera toxin, stimulates Na-H exchange. Because Na-H exchange activity was absent in control Cl⁻-depleted fibers, the Cl⁻-dependent step is at or before the G protein in the shrinkage signal-transduction pathway. The stimulation by AlF₃ indicates that the G protein is a heterotrimeric G protein.     

Induction of Exocytosis from Permeabilized Mast Cells by the Guanosine Triphosphatases Rac and Cdc42

We applied recombinant forms of the Rho-related small guanosine triphosphatases (GTPases) Rac2 and Cdc42/G25K to permeabilized mast cells to test their ability to regulate exocytotic secretion. Mast cells permeabilized with streptolysin-O leak soluble (cytosol) proteins over a period of 5 min and become refractory to stimulation by Ca²⁺ and guanosine triphosphate (GTP)γS over about 20–30 min. This loss of sensitivity is likely to be due to loss of key regulatory proteins that are normally tethered at intracellular locations. Exogenous proteins that retard this loss of sensitivity to stimulation may be similar, if not identical, to those secretory regulators that are lost. Recombinant Rac and Cdc42/G25K, preactivated by binding GTPγS, retard the loss of sensitivity (run-down) and, more importantly, enable secretion to be stimulated by Ca²⁺ alone. Investigation of the concentration dependence of each of these two GTPases applied individually to the permeabilized cells, and of Cdc42/G25K applied in the presence of an optimal concentration of Rac2, has provided evidence for a shared effector pathway and also a second effector pathway activated by Cdc42/G25K alone. Dominant negative mutant (N17) forms of Rac2 and Cdc42/G25K inhibit secretion induced by Ca²⁺ and GTPγS. Our data suggest that Rac2 and Cdc42 should be considered as candidates for G_E, GTPases that mediate exocytosis in cells of hematopoeitic origin.     

Characterization of nigericin-stimulated ATPase from sealed microsomal vesicles of tobacco callus

To understand the function and membrane origin of ionophore-stimulated ATPases, the activity of nigericin-stimulated ATPase was characterized from a low-density microsomal fraction containing sealed vesicles of autonomous tobacco (Nicotiana tabacum Linnaeous cv. Wisconsin no. 38) callus. The properties of KCl-stimulated, Mg-requiring ATPases (KCl-Mg,ATPase) were similar in the absence or presence of nigericin. Nigericin (or gramicidin) stimulation of a KCl-Mg,ATPase activity was optimum at pH 6.5 to 7.0. The enzyme was inhibited completely by N,N′-dicyclohexylcarbodiimide (10 μm), tributyltin (5 μm), and partially by vanadate (200 μm), but it was insensitive to fusicoccin and mitochondrial ATPase inhibitors, such as azide (1 mm) and oligomycin (5 μg/ml). The ATPase was more sensitive to anions than cations. Cations stimulated ATPase activity with a selectivity sequence of NH₄⁺ > K⁺, Rb⁺, Cs⁺, Na⁺, Li⁺ > Tris⁺. Anions stimulated Mg, ATPase activity with a decreasing sequence of Cl⁻ = acetate > SO₄²⁻ > benzene sulfonate > NO₃⁻. The anion stimulation was caused partly by dissipation of the electrical potential (interior positive) by permeant anions and partly by a specific ionic effect. Plant membranes had at least two classes of nigericin-stimulated ATPases: one sensitive and one insensitive to vanadate. Many of the properties of the nigericin-sensitive, salt-stimulated Mg,ATPase were similar to a vanadate-sensitive plasma membrane ATPase of plant tissues, yet other properties (anion stimulation and vanadate insensitivity) resembled those of a tonoplast ATPase. These results support the idea that nigericin-stimulated ATPases are mainly electrogenic H⁺ pumps originated in part from the plasma membrane and in part from other nonmitochondrial membranes, such as the tonoplast.     

Agonist-induced Ca2+ Sensitization in Smooth Muscle: REDUNDANCY OF RHO GUANINE NUCLEOTIDE EXCHANGE FACTORS (RhoGEFs) AND RESPONSE KINETICS,A CAGED COMPOUND STUDY*

Many agonists, acting through G-protein-coupled receptors and Gα subunits of the heterotrimeric G-proteins, induce contraction of smooth muscle through an increase of [Ca²⁺]_i as well as activation of the RhoA/RhoA-activated kinase pathway that amplifies the contractile force, a phenomenon known as Ca²⁺ sensitization. Gα_12/13 subunits are known to activate the regulator of G-protein signaling-like family of guanine nucleotide exchange factors (RhoGEFs), which includes PDZ-RhoGEF (PRG) and leukemia-associated RhoGEF (LARG). However, their contributions to Ca²⁺-sensitized force are not well understood. Using permeabilized blood vessels from PRG(−/−) mice and a new method to silence LARG in organ-cultured blood vessels, we show that both RhoGEFs are activated by the physiologically and pathophysiologically important thromboxane A₂ and endothelin-1 receptors. The co-activation is the result of direct and independent activation of both RhoGEFs as well as their co-recruitment due to heterodimerization. The isolated recombinant C-terminal domain of PRG, which is responsible for heterodimerization with LARG, strongly inhibited Ca²⁺-sensitized force. We used photolysis of caged phenylephrine, caged guanosine 5′-O-(thiotriphosphate) (GTPγS) in solution, and caged GTPγS or caged GTP loaded on the RhoA·RhoGDI complex to show that the recruitment and activation of RhoGEFs is the cause of a significant time lag between the initial Ca²⁺ transient and phasic force components and the onset of Ca²⁺-sensitized force.     

Inhibition of Endosome Fusion by Wortmannin Persists in the Presence of Activated rab5

Rab5-dependent endosome fusion is sensitive to the phosphoinositide 3-kinase inhibitor, wortmannin. It has been proposed that phosphoinositide 3-kinase activity may be required for activation of rab5 by influencing its nucleotide cycle such as to promote its active GTP state. In this report we demonstrate that endosome fusion remains sensitive to wortmannin despite preloading of endosomes with stimulatory levels of a GTPase-defective mutant rab5^Q79L or of a xanthosine triphosphate-binding mutant, rab5^D136N, in the presence of the nonhydrolysable analogue XTPγS. These results suggest that activation of rab5 cannot be the principal function of the wortmannin-sensitive factor on the endosome fusion pathway. This result is extrapolated to all GTPases by demonstrating that endosome fusion remains wortmannin sensitive despite prior incubation with the nonhydrolysable nucleotide analogue GTPγS. Consistent with these results, direct measurement of clathrin-coated vesicle-stimulated nucleotide dissociation from exogenous rab5 was insensitive to the presence of wortmannin. A large excess of rab5^Q79L, beyond levels required for maximal stimulation of the fusion assay, afforded protection against wortmannin inhibition, and partial protection was also observed with an excess of wild-type rab5 independent of GTPγS.     

Potential-dependent anion transport in tonoplast vesicles from oat roots

Potential-dependent anion movement into tonoplast vesicles from oat roots (Avena sativa L. var Lang) was monitored as dissipation of membrane potentials (Δψ) using the fluorescence probe Oxonol V. The potentials (positive inside) were generated with the H⁺-pumping pyrophosphatase, which is K⁺ stimulated and anion insensitive. The relative rate of ΔΨ dissipation by anions was used to estimate the relative permeabilities of the anions. In decreasing order they were: SCN⁻ (100) > NO₃⁻ (72) = Cl⁻ (70) > Br⁻ (62) > SO₄²⁻ (5) = H₂PO₄⁻ (5) > malate (3) = acetate (3) > iminodiacetate (2). Kinetic studies showed that the rate of Δψ dissipation by Cl⁻ and NO₃⁻, but not by SCN⁻, was saturable. The K_m values for Cl⁻ and NO₃⁻ uptake were about 2.3 and 5 millimolar, respectively, suggesting these anions move into the vacuole through proteinaceous porters. In contrast to a H⁺-coupled Cl⁻ transporter on the same vesicles, the potential-dependent Cl⁻ transport was insensitive to 4,4′-diisothiocyano-2,2′-stilbene disulfonate. These results suggest the existence of at least two different mechanisms for Cl⁻ transport in these vesicles. The potentials generated by the H⁺-translocating ATPase and H⁺-pyrophosphatase were nonadditive, giving support to the model that both pumps are on tonoplast vesicles. No evidence for a putative Cl⁻ conductance on the anion-sensitive H⁺-ATPase was found.     

Spatiotemporal dynamics of the COPI vesicle machinery

Assembly of the coat protein I (COPI) vesicle coat is controlled by the small GTPase ADP ribosylation factor 1 (ARF1) and its GTPase-activating protein, ARFGAP1. Here, we investigate the diffusional behaviours of coatomer, the main component of the coat, and also those of ARF1 and ARFGAP1. Using fluorescence-correlation spectroscopy, we found that most ARF1 and ARFGAP1 molecules are highly mobile in the cytosol (diffusion constant D ≈ 15 μm² s⁻¹), whereas coatomer diffuses 5–10 times more slowly than expected (D ≈ 1 μm² s⁻¹). This slow diffusion causes diffusion-limited binding kinetics to Golgi membranes, which, in FRAP (fluorescence recovery after photobleaching) experiments, translates into a twofold slower binding rate. The addition of aluminium fluoride locks coatomer onto Golgi membranes and also decreases the binding kinetics of both ARF1 and ARFGAP1, suggesting that these proteins function in concert to mediate sorting and vesicle formation.     

Regulation of Actin Polymerization in Cell-free Systems by GTPγS and Cdc42

We have established a cell-free system to investigate pathways that regulate actin polymerization. Addition of GTPγS to lysates of polymorphonuclear leukocytes (PMNs) or Dictyostelium discoideum amoeba induced formation of filamentous actin. The GTPγS appeared to act via a small G-protein, since it was active in lysates ofD. discoideum mutants missing either the α₂- or β-subunit of the heterotrimeric G-protein required for chemoattractant-induced actin polymerization in living cells. Furthermore, recombinant Cdc42, but not Rho or Rac, induced polymerization in the cell-free system. The Cdc42-induced increase in filamentous actin required GTPγS binding and was inhibited by a fragment of the enzyme PAK1 that binds Cdc42.

In a high speed supernatant, GTPγS alone was ineffective, but GTPγS-loaded Cdc42 induced actin polymerization, suggesting that the response was limited by guanine nucleotide exchange. Stimulating exchange by chelating magnesium, by adding acidic phospholipids, or by adding the exchange factors Cdc24 or Dbl restored the ability of GTPγS to induce polymerization. The stimulation of actin polymerization did not correlate with PIP₂ synthesis.

     

Kinetic Parameters of Denitrification in a River Continuum

Kinetic parameters for nitrate reduction in intact sediment cores were investigated by using the acetylene blockage method at five sites along the Swale-Ouse river system in northeastern England, including a highly polluted tributary, R. Wiske. The denitrification rate in sediment containing added nitrate exhibited a Michaelis-Menten-type curve. The concentration of nitrate for half-maximal activity (K_map) by denitrifying bacteria increased on passing downstream from 13.1 to 90.4 μM in the main river, but it was highest (640 μM) in the Wiske. The apparent maximal rate (V_maxap) ranged between 35.8 and 324 μmol of N m⁻² h⁻¹ in the Swale-Ouse (increasing upstream to downstream), but it was highest in the Wiske (1,194 μmol N m⁻² h⁻¹). A study of nitrous oxide (N₂O) production at the same time showed that rates ranged from below the detection limit (0.05 μmol of N₂O-N m⁻² h⁻¹) at the headwater site to 27 μmol of N₂O-N m⁻² h⁻¹ at the downstream site. In the Wiske the rate was up to 570 μmol of N₂O-N m⁻² h⁻¹, accounting for up to 80% of total N gas production.     

Characterization of a NO(3)-Sensitive H-ATPase from Corn Roots

When assayed in the presence of azide, NO₃⁻ was shown to be a specific inhibitor of a proton-translocating ATPase present in corn (Zea mays L. cv WF9 × M017) root microsomal membranes. The distribution of the NO₃⁻-sensitive ATPase on sucrose gradients and its general characteristics are similar to those previously reported for the anion-stimulated H⁺-ATPase of corn roots believed to be of tonoplast origin. An ATPase inhibited by 20 μm vanadate and insensitive to molybdate was also identified in corn root microsomal membranes which could be largely separated from the NO₃⁻-sensitive ATPase on sucrose gradients and is believed to be of plasma membrane origin. Inasmuch as both ATPase most likely catalyze the efflux of H⁺ from the cytoplasm, our objective was to characterize and compare the properties of both ATPases under identical experimental conditions. The vanadate-sensitive ATPase was stimulated by cations (K⁺ > NH₄⁺ > Rb⁺ > Cs⁺ > Li⁺ > Na⁺ > choline⁺) whereas the NO₃⁻-sensitive ATPase was stimulated by anions (Cl⁻ > Br⁻ > C₂H₃O₂⁻ > SO₄²⁻ > I⁻ > HCO₃⁻ > SCN⁻). Both ATPases required divalent cations. However, the order of preference for the NO₃⁻-sensitive ATPase (Mn²⁺ > Mg²⁺ > Co²⁺ > Ca²⁺ > Zn²⁺) differed from that of the vanadate-sensitive ATPase (Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺). The vanadate-sensitive ATPase required higher concentrations of Mg:ATP for full activity than did the NO₃⁻-sensitive ATPase. The kinetics for Mg:ATP were complex for the vanadate-sensitive ATPase, indicating positive cooperativity, but were simple for the NO₃⁻-sensitive ATPase. Both ATPases exhibited similar temperature and pH optima (pH 6.5). The NO₃⁻-sensitive ATPase was stimulated by gramicidin and was associated with NO₃⁻-inhibitable H⁺ transport measured as quenching of quinacrine fluorescence. It was insensitive to molybdate, azide, and vanadate, but exhibited slight sensitivity to ethyl-3-(3-dimethylaminopropyl carbodiimide) and mersalyl. Overall, these results indicate several properties which distinguish these two ATPases and suggest that under defined conditions NO₃⁻-sensitive ATPase activity may be used as a quantitative marker for those membranes identified tentatively as tonoplast in mixed or nonpurified membrane fractions. We feel that NO₃⁻ sensitivity is a better criterion by which to identify this ATPase than either Cl⁻ stimulation or H⁺ transport because it is less ambiguous. It is also useful in identifying the enzyme following solubilization.     

Activation status-coupled transient S acylation determines membrane partitioning of a plant Rho-related GTPase

ROPs or RACs are plant Rho-related GTPases implicated in the regulation of a multitude of signaling pathways that function at the plasma membrane by virtue of posttranslational lipid modifications. The relationship between ROP activation status and membrane localization has not been established. Here we demonstrate that endogenous ROPs, as well as a transgenic His₆-green fluorescent protein (GFP)-AtROP6 fusion protein, were partitioned between Triton X-100-soluble and -insoluble membranes. In contrast, an activated His₆-GFP-Atrop6^CA mutant protein accumulated exclusively in detergent-resistant membranes. GDP induced accumulation of ROPs in Triton-soluble membranes, whereas GTPγS induced accumulation of ROPs in detergent-resistant membranes. Recombinant wild-type and constitutively active AtROP6 isoforms were purified from Arabidopsis plants, and their lipids were cleaved and analyzed by gas chromatography-coupled mass spectrometry. In Triton-soluble membranes, wild-type AtROP6 was only prenylated, primarily by geranylgeranyl. The activated AtROP6 that accumulated in detergent-resistant membranes was modified by prenyl and acyl lipids. The acyl lipids were identified as palmitic and stearic acids. In agreement, activated His₆-GFP-Atrop6^CAmS¹⁵⁶ in which cysteine¹⁵⁶ was mutated into serine accumulated in Triton-soluble membranes. These findings show that upon GTP binding and activation, AtROP6 and possibly other ROPs are transiently S acylated, which induces their partitioning into detergent-resistant membranes.     

The Impact of Alpha-Syntrophin Deletion on the Changes in Tissue Structure and Extracellular Diffusion Associated with Cell Swelling under Physiological and Pathological Conditions

Aquaporin-4 (AQP4) is the primary cellular water channel in the brain and is abundantly expressed by astrocytes along the blood-brain barrier and brain-cerebrospinal fluid interfaces. Water transport via AQP4 contributes to the activity-dependent volume changes of the extracellular space (ECS), which affect extracellular solute concentrations and neuronal excitability. AQP4 is anchored by α-syntrophin (α-syn), the deletion of which leads to reduced AQP4 levels in perivascular and subpial membranes. We used the real-time iontophoretic method and/or diffusion-weighted magnetic resonance imaging to clarify the impact of α-syn deletion on astrocyte morphology and changes in extracellular diffusion associated with cell swelling in vitro and in vivo. In mice lacking α-syn, we found higher resting values of the apparent diffusion coefficient of water (ADC_W) and the extracellular volume fraction (α). No significant differences in tortuosity (λ) or non-specific uptake (k′), were found between α-syn-negative (α-syn −/−) and α-syn-positive (α-syn +/+) mice. The deletion of α-syn resulted in a significantly smaller relative decrease in α observed during elevated K⁺ (10 mM) and severe hypotonic stress (−100 mOsmol/l), but not during mild hypotonic stress (−50 mOsmol/l). After the induction of terminal ischemia/anoxia, the final values of ADC_W as well as of the ECS volume fraction α indicate milder cell swelling in α-syn −/− in comparison with α-syn +/+ mice. Shortly after terminal ischemia/anoxia induction, the onset of a steep rise in the extracellular potassium concentration and an increase in λ was faster in α-syn −/− mice, but the final values did not differ between α-syn −/− and α-syn +/+ mice. This study reveals that water transport through AQP4 channels enhances and accelerates astrocyte swelling. The substantially altered ECS diffusion parameters will likely affect the movement of neuroactive substances and/or trophic factors, which in turn may modulate the extent of tissue damage and/or drug distribution.     

Thermotolerance of Leaf Discs from Four Isoprene-Emitting Species Is Not Enhanced by Exposure to Exogenous Isoprene

The effects of exogenously supplied isoprene on chlorophyll fluorescence characteristics were examined in leaf discs of four isoprene-emitting plant species, kudzu (Pueraria lobata [Willd.] Ohwi.), velvet bean (Mucuna sp.), quaking aspen (Populus tremuloides Michx.), and pussy willow (Salix discolor Muhl). Isoprene, supplied to the leaves at either 18 μL L⁻¹ in compressed air or 21 μL L⁻¹ in N₂, had no effect on the temperature at which minimal fluorescence exhibited an upward inflection during controlled increases in leaf-disc temperature. During exposure to 1008 μmol photons m⁻² s⁻¹ in an N₂ atmosphere, 21 μL L⁻¹ isoprene had no effect on the thermally induced inflection of steady-state fluorescence. The maximum quantum efficiency of photosystem II photochemistry decreased sharply as leaf-disc temperature was increased; however, this decrease was unaffected by exposure of leaf discs to 21 μL L⁻¹ isoprene. Therefore, there were no discernible effects of isoprene on the occurrence of symptoms of high-temperature damage to thylakoid membranes. Our data do not support the hypothesis that isoprene enhances leaf thermotolerance.     

Development of Functional Human NK Cells in an Immunodeficient Mouse Model with the Ability to Provide Protection against Tumor Challenge

Studies of human NK cells and their role in tumor suppression have largely been restricted to in vitro experiments which lack the complexity of whole organisms, or mouse models which differ significantly from humans. In this study we showed that, in contrast to C57BL/6 Rag2^−/−/γ_c ^−/− and NOD/Scid mice, newborn BALB/c Rag2^−/−/γ_c ^−/− mice can support the development of human NK cells and CD56+ T cells after intrahepatic injection with hematopoietic stem cells. The human CD56⁺ cells in BALB/c Rag2^−/−/γ_c ^−/− mice were able to produce IFN-γ in response to human IL-15 and polyI:C. NK cells from reconstituted Rag2^−/−/γ_c ^−/− mice were also able to kill and inhibit the growth of K562 cells in vitro and were able to produce IFN-γ in response to stimulation with K562 cells. In vivo, reconstituted Rag2^−/−/γ_c ^−/− mice had higher survival rates after K562 challenge compared to non-reconstituted Rag2^−/−/γ_c ^−/− mice and were able to control tumor burden in various organs. Reconstituted Rag2^−/−/γ_c ^−/− mice represent a model in which functional human NK and CD56+ T cells can develop from stem cells and can thus be used to study human disease in a more clinically relevant environment.     

GAR22β regulates cell migration,sperm motility,and axoneme structure

Spatiotemporal cytoskeleton remodeling is pivotal for cell adhesion and migration. Here we investigated the function of Gas2-related protein on chromosome 22 (GAR22β), a poorly characterized protein that interacts with actin and microtubules. Primary and immortalized GAR22β^−/− Sertoli cells moved faster than wild-type cells. In addition, GAR22β^−/− cells showed a more prominent focal adhesion turnover. GAR22β overexpression or its reexpression in GAR22β^−/− cells reduced cell motility and focal adhesion turnover. GAR22β–actin interaction was stronger than GAR22β–microtubule interaction, resulting in GAR22β localization and dynamics that mirrored those of the actin cytoskeleton. Mechanistically, GAR22β interacted with the regulator of microtubule dynamics end-binding protein 1 (EB1) via a novel noncanonical amino acid sequence, and this GAR22β–EB1 interaction was required for the ability of GAR22β to modulate cell motility. We found that GAR22β is highly expressed in mouse testes, and its absence resulted in reduced spermatozoa generation, lower actin levels in testes, and impaired motility and ultrastructural disorganization of spermatozoa. Collectively our findings identify GAR22β as a novel regulator of cell adhesion and migration and provide a foundation for understanding the molecular basis of diverse cytoskeleton-dependent processes.     

Purification and partial characterization of a membrane-associated lipoxygenase in tomato fruit

Membrane-associated lipoxygenase from green tomato (Lycopersicon esculentum L. cv Caruso) fruit has been purified 49-fold to a specific activity of 8.3 μmol·min⁻¹·mg⁻¹ of protein by solubilization of microsomal membranes with Triton X-100, followed by anion- exchange and size-exclusion chromatography. The apparent molecular mass of the enzyme was estimated to be 97 and 102 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, respectively. The purified membrane lipoxygenase preparation consisted of a single major band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which cross-reacts with immunoserum raised against soluble soybean lipoxygenase 1. It has a pH optimum of 6.5, an apparent K_m of 6.2 μm, and V_max of 103. μmol·min⁻¹·mg⁻¹ of protein with linoleic acid as substrate. Corresponding values for the partially purified soluble lipoxygenase from tomato are 3.8 μm and 1.3 μmol·min⁻¹·mg⁻¹ of protein, respectively. Thus, the membrane-associated enzyme is kinetically distinguishable from its soluble counterpart. Sucrose density gradient fractionation of the isolated membranes indicated that the membrane-associated lipoxygenase sediments with thylakoids. A lipoxygenase band with a corresponding apparent mol wt of 97,000 was identified immunologically in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of purified thylakoids prepared from intact chloroplasts isolated from tomato leaves and fruit.     

Electrogenic transport of protons driven by the plasma membrane ATPase in membrane vesicles from radish : biochemical characterization

Mg:ATP-dependent H⁺ pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO₃⁻. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H⁺-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO₄²⁻, Cl⁻, Br⁻, NO₃⁻. ATP-dependent H⁺ pumping strictly requires Mg²⁺. It is very specific for ATP (apparent K_m 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO₄ and diethylstilbestrol as well as vanadate. Δ pH generation is specificially stimulated by K⁺ (+ 80%) and to a lesser extent by Na⁺ and choline (+28% and +14%, respectively). The characteristics of H⁺ pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.     

The Catalytic Subunit of Protein Phosphatase 1 Gamma Regulates Thrombin-Induced Murine Platelet αIIbβ3 Function

Background

Hemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin α_IIbβ₃. Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c) interacts with α_IIbβ₃, the role of PP1c in platelet reactivity is unclear.

Methodology/Principal Findings

Using γ isoform of PP1c deficient mice (PP1cγ^−/−), we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4)-activating peptide but not to adenosine diphosphate (ADP), collagen or collagen-related peptide (CRP). Thrombin-stimulated PP1cγ^−/− platelets showed decreased α_IIbβ₃ activation despite comparable levels of α_IIbβ₃, PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin α_IIbβ₃ signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cγ^−/− platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cγ^−/− mice. Phosphorylation of glycogen synthase kinase (GSK3)β-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cγ^−/− platelets by an AKT independent mechanism. Inhibition of GSK3β partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cγ^−/− platelets.

Conclusions/Significance

These studies illustrate a role for PP1cγ in maintaining GSK3β-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation.     

Fractionation of the stable isotopes of inorganic carbon by seagrasses

The δ¹³C values for seagrasses collected along the Texas Gulf Coast range from −10.9 to −11.4‰. These values are similar to the δ¹³C values of terrestrial C₄ plants, but seagrasses lack bundle sheath cells which are important in determining the δ¹³C values of C₄ plants. This work attempts to explain the reason the δ¹³C values of seagrasses resemble the δ¹³C values of C₄ plants.