Visuomotor control in continuous response time tasks across different age groups.

The goal was to examine whether visuomotor control and choice response time shared age-related developmental trajectories, and if prior computer experience played an important role in control processes. Children (6-7, 8-9, 10-11 yr.), younger adults (24 yr.) and older adults (76 yr.) performed the cursor pointing and choice response time (CRT) tasks with a computer mouse. Participants moved the mouse cursor back and forth to click two targets on the screen as fast and accurately as possible. In the CRT, based on visual stimuli, participants moved and clicked one of the three targets on the screen as fast and accurately as possible; the time between stimulus onset and clicking the correct target was recorded as the choice response time. Visuomotor performance increased with age to younger adulthood but was worse in the older adult group. CRT performance was also positively related to age among the groups of children, with scores leveling off in the young adult group. Computer experience was statistically significantly related only to visuomotor control, but not to CRT. Optimal CRT performance required only sub-optimal visuomotor control. Cognitive and sensory age declines may be related to the poorer CRT performance in the oldest age group.

ATP-binding cassette transporter A7 enhances phagocytosis of apoptotic cells and associated ERK signaling in macrophages.

The mammalian ATP-binding cassette transporters A1 and A7 (ABCA1 and -A7) show sequence similarity to CED-7, a Caenorhabditis elegans gene that mediates the clearance of apoptotic cells. Using RNA interference or gene targeting, we show that knock down of macrophage ABCA7 but not -A1 results in defective engulfment of apoptotic cells. In response to apoptotic cells, ABCA7 moves to the macrophage cell surface and colocalizes with the low-density lipoprotein receptor-related protein 1 (LRP1) in phagocytic cups. The cell surface localization of ABCA7 and LRP1 is defective in ABCA7-deficient cells. C1q is an opsonin of apoptotic cells that acts via phagocyte LRP1 to induce extracellular signal-regulated kinase (ERK) signaling. We show that ERK signaling is required for phagocytosis of apoptotic cells and that ERK phosphorylation in response to apoptotic cells or C1q is defective in ABCA7-deficient cells. These studies reveal a major role of ABCA7 and not -A1 in the clearance of apoptotic cells and therefore suggest that ABCA7 is an authentic orthologue of CED-7.

Aggregated LDL in contact with macrophages induces local increases in free cholesterol levels that regulate local actin polymerization.

OBJECTIVE: Interaction of macrophages with aggregated matrix-anchored lipoprotein deposits is an important initial step in atherogenesis. Aggregated lipoproteins require different cellular uptake processes than those used for endocytosis of monomeric lipoproteins. In this study, we tested the hypothesis that engagement of aggregated LDL (agLDL) by macrophages could lead to local increases in free cholesterol levels and that these increases in free cholesterol regulate signals that control cellular actin. METHODS AND RESULTS: AgLDL resides for prolonged periods in surface-connected compartments. Although agLDL is still extracellular, we demonstrate that an increase in free cholesterol occurs at sites of contact between agLDL and cells because of hydrolysis of agLDL-derived cholesteryl ester. This increase in free cholesterol causes enhanced actin polymerization around the agLDL. Inhibition of cholesteryl ester hydrolysis results in decreased actin polymerization. CONCLUSIONS: We describe a novel process that occurs during agLDL-macrophage interactions in which local release of free cholesterol causes local actin polymerization, promoting a pathological positive feedback loop for increased catabolism of agLDL and eventual foam cell formation.

Elevated cholesterol levels in the plasma membranes of macrophages inhibit migration by disrupting RhoA regulation.

OBJECTIVE: Atherogenesis begins as small subendothelial accumulations of foam cells that develop through unregulated uptake of modified and aggregated low-density lipoprotein (LDL). The reason why foam cells remain in the atherosclerotic plaque rather than migrating out of the area is unclear. We tested the hypothesis that elevated membrane cholesterol levels, which may result from interactions with aggregated LDL, affect macrophage migration. METHODS AND RESULTS: Cholesterol loading by incubation with cholesterol-chelated methyl-beta-cyclodextrin decreased migration of J774A.1 macrophages toward complement 5a (C5a) in transwell migration assays, even though cholesterol-loaded macrophages responded to a bath application of C5a. In a micropipette polarization assay, cholesterol-loaded cells polarized toward a C5a gradient. In a transwell migration assay, cholesterol-loaded cells extended lamellae through the filter pores but were unable to translocate their cell bodies. Cholesterol loading decreased both the cellular levels of GTP-bound active RhoA and the phosphorylation of myosin light chain. Expression of constitutively active RhoA largely prevented the inhibition of cell migration by cholesterol loading. CONCLUSIONS: These results suggest that increases in plasma membrane cholesterol content alter RhoA activation, resulting in inhibition of cell migration. These findings provide one possible explanation for the retention of foam cells in atherosclerotic lesions.

Expression and characterization of Arabidopsis phospholipase Dgamma2.

The phospholipase D (PLD) family of Arabidopsis thaliana has 12 identified members, including three highly homologous PLDgammas. The enzymatic and molecular properties of PLDgamma2 were characterized and compared with those of PLDgamma1. Two variants of PLDgamma2 cDNAs, designated PLDgamma2a and PLDgamma2b, were isolated, and they differ in the presence of a 96-nucleotide fragment at the beginning of the open reading frame. Catalytically active PLDgamma2a was expressed in E. coli. PLDgamma2a and gamma1 both require phosphatidylinositol 4,5-bisphosphate (PIP(2)) and calcium for activity, but they differ in the effect of PIP(2) and Triton X-100 on their activities. While Triton X-100 could greatly activate PLDgamma1 activity and served only as a neutral diluent in the substrate vesicles, it totally abolished PLDgamma2a activity and prohibited any stimulation effect from PIP(2.) PLDgamma2a misses one of the basic, PIP(2)-interacting residues, which may weaken the binding of PIP(2) to PLDgamma2a. In addition, PLDgamma2 and PLDgamma1 displayed different patterns of expression in different tissues, and the transcript of PLDgamma2a differs from that of PLDgamma1 by having a longer 5'-UTR. These differences in biochemical and molecular properties suggest that the highly homologous PLDgammas are subjected to unique regulations and might have distinguishable functions.

Elevated plasma membrane cholesterol content alters macrophage signaling and function.

OBJECTIVE: During atherogenesis, macrophages migrate into the subendothelial space where they ingest deposited lipoproteins, accumulate lipids, and transform into foam cells. It is unclear why these macrophages do not remove their lipid loads from the region. This study was aimed at testing the hypothesis that macrophage behavior is altered when membrane cholesterol levels are elevated, as might be the case for cells in contact with lipoproteins within atherosclerotic lesions. METHODS AND RESULTS: We examined the effects of elevating membrane cholesterol on macrophage behavior. J774 macrophages were treated with either acetylated low-density lipoprotein (ac-LDL) and ACAT inhibitor or cholesterol-chelated methyl-beta-cyclodextrin (chol-MbetaCD) to increase membrane cholesterol levels. Our results show that elevating the membrane cholesterol of J774 macrophages induced dramatic ruffling, stimulated cell spreading, and affected F-actin organization. Cellular adhesion was required for these effects, and Rac-mediated signaling pathways were involved. Additionally, 3-dimensional transwell chemotaxis assays showed that migration of J774 macrophages was significantly inhibited when membrane cholesterol levels were raised. CONCLUSIONS: These findings indicate that increased membrane cholesterol causes dramatic effects on macrophage cellular functions related to the actin cytoskeleton. They should provide new insights into the early steps of atherogenesis.

Networking of phospholipases in plant signal transduction.

Phospholipases are activated in response to various cellular and environmental cues. Their activation can affect many cellular processes through their roles in signal transduction. Recent advances in the biochemical and molecular understanding of phospholipase D (PLD) have provided insights into potential networks of PLDs and other phospholipases in plants. PLDs are a family of heterogeneous enzymes, and the activities of the multiple types of PLDs are regulated in distinctly different manners. Phosphoinositides, free fatty acids, lysophospholipids, and calcium are differential modulators of PLDs. Since these modulators are substrates, products, or downstream targets of phospholipase As and phospholipase Cs, there are many potential regulatory and metabolic interrelationships among the various PLDs and other phospholipases.

Double knockouts of phospholipases Dzeta1 and Dzeta2 in Arabidopsis affect root elongation during phosphate-limited growth but do not affect root hair patterning.

Root elongation and root hair formation are important in nutrient absorption. We found that two Arabidopsis (Arabidopsis thaliana) phospholipase Ds (PLDs), PLDzeta1 and PLDzeta2, were involved in root elongation during phosphate limitation. PLDzeta1 and PLDzeta2 are structurally different from the majority of plant PLDs by having phox and pleckstrin homology domains. Both PLDzetas were expressed more in roots than in other tissues. It was reported previously that inducible suppression or inducible overexpression of PLDzeta1 affected root hair patterning. However, gene knockouts of PLDzeta1, PLDzeta2, or the double knockout of PLDzeta1 and PLDzeta2 showed no effect on root hair formation. The expression of PLDzetas increased in response to phosphate limitation. The elongation of primary roots in PLDzeta1 and PLDzeta2 double knockout mutants was slower than that of wild type and single knockout mutants. The loss of PLDzeta2, but not PLDzeta1, led to a decreased accumulation of phosphatidic acid in roots under phosphate-limited conditions. These results indicate that PLDzeta1 and PLDzeta2 play a role in regulating root development in response to nutrient limitation.

The Arabidopsis phospholipase D family. Characterization of a calcium-independent and phosphatidylcholine-selective PLD zeta 1 with distinct regulatory domains.

Four types of phospholipase D (PLD), PLD alpha, beta, gamma, and delta, have been characterized in Arabidopsis, and they display different requirements for Ca(2+), phosphatidylinositol 4,5-bisphosphate (PIP(2)), substrate vesicle composition, and/or free fatty acids. However, all previously cloned plant PLDs contain a Ca(2+)-dependent phospholipid-binding C2 domain and require Ca(2+) for activity. This study documents a new type of PLD, PLD zeta 1, which is distinctively different from previously characterized PLDs. It contains at the N terminus a Phox homology domain and a pleckstrin homology domain, but not the C2 domain. A full-length cDNA for Arabidopsis PLD zeta 1 has been identified and used to express catalytically active PLD in Escherichia coli. PLD zeta 1 does not require Ca(2+) or any other divalent cation for activity. In addition, it selectively hydrolyzes phosphatidylcholine, whereas the other Arabidopsis PLDs use several phospholipids as substrates. PLD zeta 1 requires PIP(2) for activity, but unlike the PIP(2)-requiring PLD beta or gamma, phosphatidylethanolamine is not needed in substrate vesicles. These differences are described, together with a genomic analysis of 12 putative Arabidopsis PLD genes that are grouped into alpha, beta, delta, gamma, and zeta based on their gene architectures, sequence similarities, domain structures, and biochemical properties.

Kinetic analysis of Arabidopsis phospholipase Ddelta. Substrate preference and mechanism of activation by Ca2+ and phosphatidylinositol 4,5-biphosphate.

Phospholipase D (PLD) is a major plant phospholipase family involved in many cellular processes such as signal transduction, membrane remodeling, and lipid degradation. Five classes of PLDs have been identified in Arabidopsis thaliana, and Ca(2+) and polyphosphoinositides have been suggested as key regulators for these enzymes. To investigate the catalysis and regulation mechanism of individual PLDs, surface-dilution kinetics studies were carried out on the newly identified PLDdelta from Arabidopsis. PLDdelta activity was dependent on both bulk concentration and surface concentration of substrate phospholipids in the Triton X-100/phospholipid mixed micelles. V(max), K(s)(A), and K(m)(B) values for PLDdelta toward phosphatidylcholine or phosphatidylethanolamine were determined; phosphatidylethanolamine was the preferred substrate. PLDdelta activity was stimulated greatly by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Maximal activation was observed at a PIP(2) molar ratio around 0.01. Kinetic analysis indicates that PIP(2) activates PLD by promoting substrate binding to the enzyme, without altering the bulk binding of the enzyme to the micelle surface. Ca(2+) is required for PLDdelta activity, and it significantly decreased the interfacial Michaelis constant K(m)(B). This indicates that Ca(2+) activates PLD by promoting the binding of phospholipid substrate to the catalytic site of the enzyme.

Phospholipase D alpha 1-derived phosphatidic acid interacts with ABI1 phosphatase 2C and regulates abscisic acid signaling.

Phospholipase D (PLD) and protein phosphatase 2C (PP2C) both play a role in mediating plant responses to abscisic acid (ABA). In this article, we show that PLD alpha 1 and its product, phosphatidic acid (PA), regulate a PP2C, ABI1, which is a negative regulator of ABA responses in Arabidopsis. Leaves from a T-DNA insertional mutant of PLD alpha 1 and PLD alpha 1-antisense plants lose more water than do wild-type plants. The stomatal closure of PLD alpha 1-null leaves is insensitive to ABA but is promoted by PA. ABA treatment promotes an increase in PA from phosphatidylcholine in wild type but not in PLD alpha 1-null cells. PLD alpha 1-derived PA binds to ABI1; the PA-ABI1 binding is demonstrated by coprecipitating PA with ABI1 from plant cells, measuring binding of PA from vesicles to ABI1, and assaying ABI1 bound to PA immobilized on a filter. Deletion and site-specific mutational analyses show that arginine 73 in ABI1 is essential for PA-ABI1 binding. PA binding decreases the phosphatase activity of ABI1. The lack of ABA-induced production of PA in PLD alpha 1-null cells results in a decrease in the association of ABI1 with the plasma membrane in response to ABA. These results indicate that PA produced by PLD alpha 1 inhibits the function of the negative regulator ABI1, thus promoting ABA signaling. The identification of ABI1 as a direct target of the lipid messenger PA provides a functional link between the two families of important signaling enzymes, PLD and PP2C.

Evolutionary conservation of physical and functional interactions between phospholipase D and actin.

Phospholipase D (PLD) enzymes from bacteria to mammals exhibit a highly conserved core structure and catalytic mechanism, but whether protein-protein interactions exhibit similar commonality is unknown. Our objective was to determine whether the physical and functional interactions of mammalian PLDs with actin are evolutionarily conserved among bacterial and plant PLDs. Highly purified bacterial and plant PLDs cosedimented with mammalian skeletal muscle alpha-actin, indicating direct interaction with F-actin. The binding of bacterial PLD to G-actin exhibited two affinity states, with dissociation constants of 1.13 pM and 0.58 microM. The effects of actin on the activities of bacterial and plant PLDs were polymerization dependent; monomeric G-actin inhibited PLD activity, whereas polymerized F-actin augmented PLD activity. Actin modulation of bacterial and plant PLDs demonstrated kinetic characteristics, efficacies, and potencies similar to those of human PLD1. Thus, physical and functional interactions between PLD and actin in PLD family members from bacteria to mammals are highly conserved throughout evolution.

The oleate-stimulated phospholipase D, PLDdelta, and phosphatidic acid decrease H2O2-induced cell death in Arabidopsis.

Hydrolysis of common membrane phospholipids occurs in response to various environmental stresses, but the control and cellular function of this hydrolysis are not fully understood. Hydrogen peroxide (H2O2) is a pivotal signaling molecule involved in various stress responses. Here, we show that the plasma membrane-bound phospholipase D, PLDdelta, is activated in response to H2O2 and that the resulting phosphatidic acid (PA) functions to decrease H2O2-promoted programmed cell death. The Arabidopsis genome has 12 PLD genes, and knockout of PLDdelta abolishes specifically the oleate-stimulated PLD activity. H2O2 treatment of Arabidopsis cells activates PLD enzyme activity, and ablation of PLDdelta abolishes that activation. PLDdelta-null cells display increased sensitivity to H2O2-induced cell death. The addition of PA to PLDdelta-null cells mitigates the H2O2 effect, whereas suppression of the H2O2-induced PA formation in wild-type cells increases the effect. PLDdelta-ablated plants exhibit increased susceptibility to stress. These results demonstrate that activation of oleate-stimulated PLDdelta constitutes an important step in the plant response to H2O2 and increasing plant stress tolerance.