BackgroundRecent evidence indicates that in addition to the T-cell receptor, microclustering is an important mechanism for the activation of the B-cell receptor and the mast cell Fcε-receptor. In macrophages and neutrophils, particles opsonized with immunoglobulin G (IgG) antibodies activate the phagocytic Fcγ-receptor (FcγR) leading to rearrangements of the actin cytoskeleton. The purpose of this study was to establish a system for high-resolution imaging of FcγR microclustering dynamics and the recruitment of the downstream signaling machinery to these microclusters.MethodsWe developed a supported lipid bilayer platform with incorporated antibodies on its surface to study the formation and maturation of FcγR signaling complexes in macrophages. Time-lapse multicolor total internal reflection microscopy was used to capture the formation of FcγR-IgG microclusters and their assembly into signaling complexes on the plasma membrane of murine bone marrow derived macrophages.ResultsUpon antibody binding, macrophages formed FcγR-IgG complexes at the leading edge of advancing pseudopods. These complexes then moved toward the center of the cell to form a structure reminiscent of the supramolecular complex observed in the T-cell/antigen presenting cell immune synapse. Colocalization of signaling protein Syk with nascent clusters of antibodies indicated that phosphorylated receptor complexes underwent maturation as they trafficked toward the center of the cell. Additionally, imaging of fluorescent BtkPH domains indicated that 3′-phosphoinositides propagated laterally away from the FcγR microclusters.ConclusionWe demonstrate that surface-associated but mobile IgG induces the formation of FcγR microclusters at the pseudopod leading edge. These clusters recruit Syk and drive the production of diffusing PI(3,4,5)P3 that is coordinated with lamellar actin polymerization. Upon reaching maximal extension, FcγR microclusters depart from the leading edge and are transported to the center of the cellular contact region to form a synapse-like structure, analogous to the process observed for T-cell receptors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-016-0143-2) contains supplementary material, which is available to authorized users.

Arginine-vasopressin (AVP) plays a major role in maintaining cardiovascular function and related pathologies. The mechanism involved in its release into the circulation is complex and highly regulated. Recent work has implicated the purinergic receptor, P2X7R, in a role for catecholamine-enhanced AVP release in the rat hypothalamic-neurohypophysial (NH) system. However, the site of P2X7R action in this endocrine system and whether or not it directly mediates release in secretory neurons have not been determined. We hypothesized that the P2X7R is expressed and mediates AVP release in NH terminals. P2X7R function was first examined by patch-clamp recordings in isolated NH terminals. Results revealed that subpopulations of isolated terminals displayed either high ATP-sensitivity or low ATP-sensitivity, the latter of which was characteristic of the rat P2X7R. Additional recordings showed that terminals showing sensitivity to the P2X7R-selective agonist, BzATP, were further inhibited by P2X7R selective antagonists, AZ10606120 and brilliant blue-G. In confocal micrographs from isolated terminals of the NH showed that P2X7R-immunoreactivity was localized in the plasma membranes. Lastly, the role of P2X7R on AVP release was tested. Our results showed that BzATP evoked sustained AVP release in NH terminals, which was inhibited by AZ10606120. Taken together, our data lead us to conclude that the P2X7R is expressed in NH terminals and corroborates its role in AVP secretion.

are millimolar divalent cations present in the extracellular milieu, it is likely that most extracellular ATP released from synaptic vesicles is chelated by divalent. We found that some subtypes of P2X receptors can be activated by both free and divalent-bound ATP, while others can only be efficiently activated by free ATP. This subtype specific activation by different forms of ATP parallels the pharmacological sensitivity to other agonists and antagonists, pointing to the existence of two distinct classes of ligand binding pockets. We are currently examining which forms of ATP activate heteromeric P2X receptor channels formed by subunits with different sensitivity to divalent-bound ATP.

P2X7R expression in neurons is controversial; however, studies suggest that it may play a role in hormone release in the hypothalamic NH system (HNS). We examined this in isolated NH terminals (NHT). Application of the P2X7R agonist, BzATP, to NHT resulted in increased levels of sustained AVP release, compared with ATP. Further experiments showed that the sustained release was inhibited by the P2X7R antagonist brilliant blue‐G (BBG). Confocal microscopy showed P2X7R‐immunoreactivity (IR) in slice sections and in isolated terminals of the NH. However, the cell bodies in the hypothalamus that project to the NH were devoid of P2X7R‐IR. Patch clamp studies revealed that subpopulations of NHT displayed differential sensitivity to ATP in evoking ion currents. One population showed a pEC50 of 5.2 ± 0.1 M and the second showed a pEC50 of 3.9 ± 0.2 M (T‐test, p< 0.0002, n=5–6), the latter being consistent with P2X7R. In low divalent cationic media the BzATP, gave a pEC50 of 5.6 ± 0.15 and pretreatment with 10 and 300 nM BBG inhibited currents by 51.8 ± 13.1 and 12.3 ± 4.2 percent of control, respectively (1 way ANOVA, P < 0.0001, n=5). The poor sensitivity to ATP and the marked inhibition of BzATP‐mediated current by BBG are hallmarks of P2X7R function. These results lead us to conclude that P2X7R is present in NHT and play a functional role in hormone release in the HNS. (Study supported by NIH grant NS29470 to JRL)