High affinity IgE receptors (FcεRI) on mast cells form a “synapse” when presented with mobile, bilayer incorporated antigen. Here, we show that receptor reorganization within the contacting mast cell membrane is markedly different upon binding of mobile and immobilized ligands. Rat basophilic leukemia mast cells (RBL-2H3) primed with fluorescent anti-DNP IgE were engaged by surfaces presenting either bilayer-incorporated, monovalent DNP-lipid (mobile ligand) or chemically crosslinked, multivalent DNP (immobilized ligand). Total internal reflection fluorescence imaging and electron microscopy methods were used to visualize receptor reorganization at the contact site. The spatial relationships of FcεRI to other cellular components at the synapse, such as actin, cholesterol and LAT, were also analyzed. Stimulation of mast cells with immobilized polyvalent ligand resulted in typical levels of degranulation. Remarkably, degranulation also followed interaction of mast cells with bilayers presenting mobile, monovalent ligand. Receptors engaged with mobile ligand coalesce into large, cholesterol-rich clusters that occupy the central portion of the contacting membrane. These data indicate that FcεRI crosslinking is not an obligatory step in triggering mast cell signaling and suggest that dense populations of mobile receptors are capable of initiating low level degranulation upon ligand recognition.

A new silicon detector has been developed to provide the PHENIX experiment with precise charged particle tracking at forward and backward rapidity. The Forward Silicon Vertex Tracker (FVTX) was installed in PHENIX prior to the 2012 run period of the Relativistic Heavy Ion Collider (RHIC). 1 arXiv:1311.3594v2 [physics.ins-det] 14 Feb 2014The FVTX is composed of two annular endcaps, each with four stations of silicon mini-strip sensors, covering a rapidity range of 1.2 < |η| < 2.2 that closely matches the two existing PHENIX muon arms. Each station consists of 48 individual silicon sensors, each of which contains two columns of mini-strips with 75 µm pitch in the radial direction and lengths in the φ direction varying from 3.4 mm at the inner radius to 11.5 mm at the outer radius. The FVTX has approximately 0.54 million strips in each endcap. These are read out with FPHX chips, developed in collaboration with Fermilab, which are wire bonded directly to the mini-strips. The maximum strip occupancy reached in central Au-Au collisions is approximately 2.8%. The precision tracking provided by this device makes the identification of muons from secondary vertices away from the primary event vertex possible. The expected distance of closest approach (DCA) resolution of 200 µm or better for particles with a transverse momentum of 5 GeV/c will allow identification of muons from relatively long-lived particles, such as D and B mesons, through their broader DCA distributions.

There is considerable interest in the signaling mechanisms of immunoreceptors, especially when triggered with membrane-bound ligands. We have quantified the spatiotemporal dynamics of the redistribution of immunoglobulin E-loaded receptors (IgE-FcepsilonRI) on rat basophilic leukemia-2H3 mast cells in contact with fluid and gel-phase membranes displaying ligands for immunoglobulin E, using total internal reflection fluorescence microscopy. To clearly separate the kinetics of receptor redistribution from cell spreading, and to precisely define the initial contact time (+/-50 ms), micropipette cell manipulation was used to bring individual cells into contact with surfaces. On ligand-free surfaces, there are micron-scale heterogeneities in fluorescence that likely reflect regions of the cell that are more closely apposed to the substrate. When ligands are present, receptor clusters form with this same size scale. The initial rate of accumulation of receptors into the clusters is consistent with diffusion-limited trapping with D approximately 10(-1) microm2/s. These results support the hypothesis that clusters form by diffusion to cell-surface contact regions. Over longer timescales (>10 s), individual clusters moved with both diffusive and directed motion components. The dynamics of the cluster motion is similar to the dynamics of membrane fluctuations of cells on ligand-free fluid membranes. Thus, the same cellular machinery may be responsible for both processes.

When mast cells contact a monovalent antigen-bearing fluid lipid bilayer, IgE-loaded FcεRI receptors aggregate at contact points and trigger degranulation and the release of immune activators. We used two-color total internal reflection fluorescence microscopy and single-particle tracking to show that most fluorescently labeled receptor complexes diffuse freely within these micron-size clusters, with a diffusion coefficient comparable to free receptors in resting cells. At later times, when the small clusters coalesce to form larger patches, receptors diffuse even more rapidly. In all cases, Monte Carlo diffusion simulations ensured that the tracking results were free of bias, and distinguished biological from statistical variation. These results show the diversity in receptor mobility in mast cells, demonstrating at least three distinct states of receptor diffusivity.

Superparamagnetic iron oxide (Fe3O4) and highly anisotropic barium hexaferrite (BaFe12O19) nanoparticles were coated with an anti-inflammatory drug and magnetically transported through mucus produced by primary human airway epithelial cells. Using wet planetary ball milling, dl-2-amino-3-phosphonopropionic acid-coated BaFe12O19 nano-particles (BaNPs) of 1–100 nm in diameter were prepared in water. BaNPs and conventional 20–30-nm Fe3O4 nanoparticles (FeNPs) were then encased in a polymer (PLGA) loaded with dexamethasone (Dex) and tagged for imaging. PLGA-Dex-coated BaNPs and FeNPs were characterized using dynamic light scattering (DLS), transmission electron microscopy (TEM), and superconducting quantum interference device (SQUID) magnetometry. Both PLGA-Dex-coated BaNPs and FeNPs were transferred to the surface of a ~100-μm thick mucus layer of air-liquid interface cultured primary normal human tracheobronchial epithelial (NHTE) cells. Within 30 min, the nanoparticles were pulled successfully through the mucus layer by a permanent neodymium magnet. The penetration time of the nanomedicine was monitored using confocal microscopy and tailored by varying the thickness of the PLGA-Dex coating around the particles.