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Stimulated exocytic events provide a means for physiological communication and are a hallmark of the mast cell-mediated allergic response. In mast cells these processes are triggered by antigen crosslinking of IgE bound to its high-affinity receptor, FcϵRI, on the cell surface. Here we use the endosomal v-SNARE VAMP8, and the lysosomal hydrolase β-hexosaminidase (β-Hex), each C-terminally fused to super-ecliptic pHluorin, to monitor stimulated exocytosis. Using these pHluorin-tagged constructs, we monitor stimulated exocytosis by fluorimetry and visualize individual exocytic events with total internal reflection (TIRF) microscopy. Similar to constitutive recycling endosome (RE) trafficking, we find that stimulated RE exocytosis, monitored by VAMP8, is attenuated by expression of dominant negative (S25N) Rab11. Stimulated β-Hex exocytosis is also reduced in the presence of S25N Rab11, suggesting that expression of this mutant broadly impacts exocytosis. Interestingly, pretreatment with inhibitors of actin polymerization, cytochalasin D or latrunculin A, substantially restores both RE and lysosome exocytosis in cells expressing S25N Rab11. Conversely, stabilizing F-actin with jasplakinolide inhibits antigen-stimulated exocytosis but is not additive with S25N Rab11-mediated inhibition, suggesting that these reagents inhibit related processes. Together, our results suggest that Rab11 participates in the regulation necessary for depolymerization of the actin cytoskeleton during stimulated exocytosis in mast cells.

Electroactive nanofibers decorated with α,ω-bi-(dinitrophenylcaproic acid), abbreviated as DNP, have been prepared by electrospinning a solution of α,ω-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)], polystyrene and single-walled carbon nanotubes (SWCNTs). The α,ω-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethyleneoxide)] polymers were synthesized by living anionic polymerization and subsequent esterification to obtain the DNP-functionalized polymers. The nanofibers (300 nm) were electrospun onto a silicon wafer substrate at a voltage of 10 kV using dimethylformamide and chlorobenzene as solvents. The nanofibers were characterized by Raman spectroscopy, fluorescence microscopy, scanning electron microscopy (SEM), atomic force microscopy (AFM) and optical microscopy. The DNP groups, therein, were tethered to the nanofibers via oligo(oxyethylene) spacers. The DNP groups decorating the nanofibers were capable of specifically binding with anti-DNP immunoglobulin E (IgE). The binding specificity of nanofibers containing 1% SWCNTs with anti-DNP IgE was studied via fluorescently labeled (Fluorescein IsoThioCyanate) FITC-IgE. Electronic activity of the nanofibers was studied by I–V plots from Kelvin sensing. The I–V plots before sensitizing with IgE differed from the I–V plots after binding with IgE. The results of the studies suggest the possibility of developing functional nanofibers as the active component in biosensors. Copyright © 2013 John Wiley & Sons, Ltd.

Binding of fluorescein isothiocyanate (FITC)-conjugated cholera toxin B subunit to ganglioside GM1 on RBL-2H3 cells at 37 °C results in labeling of the plasma membrane as well as a pool of perinuclear intracellular membranes identified as the endosomal recycling compartment. Antigen-mediated activation of IgE receptor signaling causes rapid, sustained outward trafficking of these labeled endosomes, that is monitored as an increase in FITC fluorescence due to relief of quenching in the acidic endosomes upon delivery to the plasma membrane. Stimulation of this process depends on the integrity of cholesterol-dependent lipid rafts and occurs in response to Ca2+-mobilizing thapsigargin as well as antigen. Inhibitors of some early signaling enzymes stimulated by FceRI, including Syk tyrosine kinase and phosphoinositide 3-kinase, have little or no effect on this trafficking response. Other signaling pathways, including activation of phospholipase C and Ca2+ influx, do not appear to be necessary for the initiation of the outward trafficking response, but they contribute to maintaining the sustained phase of this process. Consistent with this, antigen-stimulated ruffles are labeled with FITC-cholera toxin B in a Ca2+-dependent manner. Thus, this trafficking response provides a mechanism by which an internal membrane pool can contribute to plasma membrane remodeling during stimulated membrane ruffling, cell motility, and phagocytosis.