Biomedical subjects

James A Barton

Publications and source records attributed to James A Barton.

4 recordsLinked to original sources

PubMed2006

Phospholipase D1 regulates phagocyte adhesion.

Adhesion is a fundamental cellular response that is essential to the physiologic processes of development, differentiation, proliferation, and motility, as well as to the pathology of inflammation, transformation, and metastasis. Adhesion of phagocytic leukocytes is a critical modulator of antimicrobial and cytotoxic functions, including the respiratory burst, secretion, and apoptosis. Because phospholipase D (PLD) is linked to several signaling pathways implicated in these processes, we tested the hypothesis that PLD regulates phagocyte adhesion. Adhesion of primary human neutrophils and monocyte-derived macrophages to fibronectin was accompanied by marked stimulation of PLD activity. Similarly, adhesion of both human (PLB, THP-1) and murine (RAW) myeloid-macrophage cell lines to fibronectin, fibrinogen, collagen, or plastic resulted in significant activation of PLD. Stimulation of PLD activity was rapid and persisted for at least 90 min. Confocal microscopy indicated that PLD1 exhibited partial colocalization with actin filaments at the adherent interface, in proximity to the focal adhesion protein, paxillin. Reductions in PLD activity by chemical inhibitors or specific short-interfering RNA-induced knockdown of PLD1 resulted in significant inhibition of phagocyte adhesion and was accompanied by reductions in total cellular F-actin. These data support the hypotheses that adhesion stimulates PLD activity, and that PLD1 regulates the initial stages of phagocyte adhesion. Stimulation of PLD activity may promote adhesion-dependent phagocyte effector responses.

Actins↗

PubMed2004

Phospholipases D1 and D2 coordinately regulate macrophage phagocytosis.

Phagocytosis is a fundamental feature of the innate immune system, required for antimicrobial defense, resolution of inflammation, and tissue remodeling. Furthermore, phagocytosis is coupled to a diverse range of cytotoxic effector mechanisms, including the respiratory burst, secretion of inflammatory mediators and Ag presentation. Phospholipase D (PLD) has been linked to the regulation of phagocytosis and subsequent effector responses, but the identity of the PLD isoform(s) involved and the molecular mechanisms of activation are unknown. We used primary human macrophages and human THP-1 promonocytes to characterize the role of PLD in phagocytosis. Macrophages, THP-1 cells, and other human myelomonocytic cells expressed both PLD1 and PLD2 proteins. Phagocytosis of complement-opsonized zymosan was associated with stimulation of the activity of both PLD1 and PLD2, as demonstrated by a novel immunoprecipitation-in vitro PLD assay. Transfection of dominant-negative PLD1 or PLD2 each inhibited the extent of phagocytosis (by 55-65%), and their combined effects were additive (reduction of 91%). PLD1 and PLD2 exhibited distinct localizations in resting macrophages and those undergoing phagocytosis, and only PLD1 localized to the phagosome membrane. The COS-7 monkey fibroblast cell line, which has been used as a heterologous system for the analysis of receptor-mediated phagocytosis, expressed PLD2 but not PLD1. These data support a model in which macrophage phagocytosis is coordinately regulated by both PLD1 and PLD2, with isoform-specific localization. Human myelomonocytic cell lines accurately model PLD-dependent signal transduction events required for phagocytosis, but the heterologous COS cell system does not.

Animals↗

PubMed2003

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.

Actins↗

PubMed2002

Regulation of phospholipase D activity by actin. Actin exerts bidirectional modulation of Mammalian phospholipase D activity in a polymerization-dependent, isoform-specific manner.

Many critical cellular processes, including proliferation, vesicle trafficking, and secretion, are regulated by both phospholipase D (PLD) and the actin microfilament system. Stimulation of human PLD1 results in its association with the detergent-insoluble actin cytoskeleton, but the molecular mechanisms and functional consequences of PLD-actin interactions remain incompletely defined. Biochemical and pharmacologic modulation of actin polymerization resulted in complex bidirectional effects on PLD activity, both in vitro and in vivo. Highly purified G-actin inhibited basal and stimulated PLD activity, whereas F-actin produced the opposite effects. Actin-induced modulation of PLD activity was independent of the activating stimulus. The efficacy and potency of the effects of actin were isoform-specific but broadly conserved among actin family members. Human betagamma-actin was only 45% as potent and 40% as efficacious as rabbit skeletal muscle alpha-actin, whereas its inhibitory profile was similar to the single actin species from the yeast, Saccharomyces cerevisiae. Use of actin polymerization-specific reagents indicated that PLD1 binds both monomeric G-actin, as well as actin filaments. These data are consistent with a model in which the physical state of the actin cytoskeleton is a critical determinant of its regulation of PLD activity.

Actins↗