Distinct Stages of Stimulated FcεRI Receptor Clustering and Immobilization Are Identified through Superresolution Imaging

Recent advances in fluorescence localization microscopy have made it possible to image chemically fixed and living cells at 20 nm lateral resolution. We apply this methodology to simultaneously record receptor organization and dynamics on the ventral surface of live RBL-2H3 mast cells undergoing antigen-mediated signaling. Cross-linking of IgE bound to FcεRI by multivalent antigen initiates mast cell activation, which leads to inflammatory responses physiologically. We quantify receptor organization and dynamics as cells are stimulated at room temperature (22°C). Within 2 min of antigen addition, receptor diffusion coefficients decrease by an order of magnitude, and single-particle trajectories are confined. Within 5 min of antigen addition, receptors organize into clusters containing ∼100 receptors with average radii of ∼70 nm. By comparing simultaneous measurements of clustering and mobility, we determine that there are two distinct stages of receptor clustering. In the first stage, which precedes stimulated Ca²⁺ mobilization, receptors slow dramatically but are not tightly clustered. In the second stage, receptors are tightly packed and confined. We find that stimulation-dependent changes in both receptor clustering and mobility can be reversed by displacing multivalent antigen with monovalent ligands, and that these changes can be modulated through enrichment or reduction in cellular cholesterol levels.     

DNA damage induced by phorbol ester-stimulated neutrophils is augmented by extracellular cofactors. Role of histidine and metals

Activated neutrophils cause extensive DNA damage in neighboring nonphagocytic cells. To determine whether compounds in the extracellular milieu participate in the DNA damage process, murine neutrophils were cocultivated with target tumor cells in media of varying composition. Using the alkaline elution assay, it was found that the level of strand breaks induced was significantly higher (2.8-fold) in complex cell culture media than in minimal phosphate-buffered saline. Addition of amino acids in general and of histidine in particular increased the level of damage nearly to that observed in complete media (2.7- and 2.1-fold, respectively). The histidine stimulation was concentration-dependent and reached a maximum at 100-400 microM. The mechanism whereby this occurred is not proven but probably derived from chelation of metals and participation in a site-specific Fenton reaction. Addition of the cell-impermeable chelator EDTA dramatically inhibited induction of strand breaks by neutrophils in complete media and prevented the enhancement of damage induced by histidine in phosphate-buffered saline. None of the effects on neutrophil-induced damage could be attributed to modulation of the oxidative burst activity of the cells (O2- and H2O2 production). Histidine also enhanced induction of strand breaks by reagent H2O2. However, EDTA had no effect or actually increased the level of damage induced by both a bolus of H2O2 and a flux of H2O2 generated by glucose oxidase. The cell-permeable chelator o-phenanthroline inhibited both neutrophil- and H2O2-induced damage. The results indicate that secondary reactions involving extracellular amino acids and metals contribute significantly to neutrophil-induced DNA damage to neighboring cells. Moreover, the data show that the mechanism whereby neutrophils induce this damage cannot be attributed solely to secretion of H2O2.     

Improvement of porphyrin cellular delivery and activity by conjugation to a carrier peptide

The chemical nuclease metalloporphyrin (manganese(III) porphyrin) can cleave DNA irreversibly and can thus constitute a potential antitumor drug. However, these molecules show low permeability to cell surface membranes. We report here the conjugation of an amphipathic carrier peptide to improve considerably its cellular delivery. The metalloporphyrin-peptide conjugate can be internalized by cells within only 5 min of incubation with a yield as high as 80%. Furthermore, the metalloporphyrin-peptide conjugate is able to cleave in vitro high or low molecular weight DNA to the same extend as metalloporphyrin alone without affecting the sequence-specific cleaving activity of the porphyrin. The conjugate is 100-fold more efficient at inducing tumor cells death than the free metalloporphyrin via a mechanism involving genomic DNA cleavage. The results are promising for further therapeutic applications with antitumor drugs such as metalloporphyrin, and also with other existing drugs by using a carrier peptide system in order to improve the cellular uptake of such molecules.