Kathrin Spendier

Formation of a Mast Cell Synapse: FcεRI Membrane Dynamics upon Binding Mobile or Immobilized Ligands on Surfaces

FcεRI on mast cells form a synapse when presented with mobile, bilayer-incorporated Ag. In this study, 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 primed with fluorescent anti-DNP IgE were engaged by surfaces presenting either bilayer-incorporated, monovalent DNP-lipid (mobile ligand), or chemically cross-linked, 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 linker for activation of T cells, 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 cross-linking 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.

Single-particle tracking of immunoglobulin E receptors (FcεRI) in micron-sized clusters and receptor patches

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.

Reaction-diffusion theory in the presence of an attractive harmonic potential.

Problems involving the capture of a moving entity by a trap occur in a variety of physical situations, the moving entity being an electron, an excitation, an atom, a molecule, a biological object such as a receptor cluster, a cell, or even an animal such as a mouse carrying an epidemic. Theoretical considerations have almost always assumed that the particle motion is translationally invariant. We study here the case when that assumption is relaxed, in that the particle is additionally subjected to a harmonic potential. This tethering to a center modifies the reaction-diffusion phenomenon. Using a Smoluchowski equation to describe the system, we carry out a study which is explicit in one dimension but can be easily extended for arbitrary dimensions. Interesting features emerge depending on the relative location of the trap, the attractive center, and the initial placement of the diffusing particle.

Distribution and Dynamics of Rat Basophilic Leukemia Immunoglobulin E Receptors (FcɛRI) on Planar Ligand-Presenting Surfaces

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.

Effects of deuterium oxide on cell growth and vesicle speed in RBL-2H3 cells

For the first time we show the effects of deuterium oxide on cell growth and vesicle transport in rat basophilic leukemia (RBL-2H3) cells. RBL-2H3 cells cultured with 15 moles/L deuterium showed decreased cell growth which was attributed to cells not doubling their DNA content. Experimental observations also showed an increase in vesicle speed for cells cultured in deuterium oxide. This increase in vesicle speed was not observed in deuterium oxide cultures treated with a microtubule-destabilizing drug, suggesting that deuterium oxide affects microtubule-dependent vesicle transport.

Investigations Into 25- and 30-

Parallel-plate capacitors constructed with 25-and 30-μm-thick alkali-free glass sheets are demonstrated in pulsed RC discharge circuit. Capacitance values ranged from ~25 to 54 nF. Capacitors were charged and discharged with an electrically triggered switch into resistive loads and were tested at 23 °C in oil up to 3.2 kV and up to 235 °C in air with a charge voltage of 350 V. Peak currents greater than 160 A were measured for multiple capacitor charge and discharge cycles for the capacitor submerged in oil. To our knowledge, this is the first demonstration of an ultrathin glass capacitor with multikilovolt charge voltage implement in a pulsed power electrical circuit. In addition, the dielectric strength of 25-μm-thick alkali-free Schott Inc. AF 32 ECO glass is investigated in Shell Diala oil at room temperature as a function of electrode area spanning a factor of 600. The results show that as electrode area increases dielectric strength significantly decreases. Implications for the decrease in dielectric strength at large dielectric areas are discussed in regard to manufacturing ultrathin glass capacitors. Dielectric strengths measured for AF 32 ECO glass are in agreement with similar compositions of alkali-free glass.

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Motivated currently by the problem of coalescence of receptor clusters in mast cells in the general subject of immune reactions, and formerly by the investigation of exciton trapping and sensitized luminescence in molecular systems and aggregates, we present analytic expressions for survival probabilities of moving entities undergoing diffusion and reaction on encounter. Results we provide cover several novel situations in simple 1-d systems as well as higher-dimensional counterparts along with a useful compendium of such expressions in chemical physics and allied fields. We also emphasize the importance of the relationship of discrete sink term analysis to continuum boundary condition studies.

-Thick Glass Capacitors at 23 °C and 235 °C and Area Dependence of Dielectric Strength of Alkali-Free Schott Inc. AF 32 ECO Glass

Controlled deposition of metal nanostructures on various substrates is highly desirable for electronics, photonics, sensing and catalysis. A laser focused beam has been used to deposit metal nanoparticles and nanostructures based on optical forces with diffraction-limited spatial resolution. We demonstrate fabrication of nanostructured silver wires, spots and wire arrays by using a confocal microscope setup, offering highly reproducible nanostructured silver growth and pattern writing. Particularly, laser-deposited silver micro-wires show adjustable electronic conductivities. Taking into accounts the superior spatial resolution and versatile pattern design, this technique is promising for a variety of applications such as microelectronics and bio-chips.

Analytic Solutions for Some Reaction-Diffusion Scenarios

Abstract Background Structured illumination microscopy (SIM) is a family of methods in optical fluorescence microscopy that can achieve both optical sectioning and super-resolution effects. SIM is a valuable method for high-resolution imaging of fixed cells or tissues labeled with conventional fluorophores, as well as for imaging the dynamics of live cells expressing fluorescent protein constructs. In SIM, one acquires a set of images with shifting illumination patterns. This set of images is subsequently treated with image analysis algorithms to produce an image with reduced out-of-focus light (optical sectioning) and/or with improved resolution (super-resolution). Findings Five complete, freely available SIM datasets are presented including raw and analyzed data. We report methods for image acquisition and analysis using open-source software along with examples of the resulting images when processed with different methods. We processed the data using established optical sectioning SIM and super-resolution SIM methods and with newer Bayesian restoration approaches that we are developing. Conclusions Various methods for SIM data acquisition and processing are actively being developed, but complete raw data from SIM experiments are not typically published. Publically available, high-quality raw data with examples of processed results will aid researchers when developing new methods in SIM. Biologists will also find interest in the high-resolution images of animal tissues and cells we acquired. All of the data were processed with SIMToolbox, an open-source and freely available software solution for SIM.

Laser-directed deposition of silver nanostructures

Biomembranes undergo extensive shape changes as they perform vital cellular functions or become diseased. To understand the mechanisms by which lipids and proteins control membrane curvature during various processes, researchers have identified and engineered many curvature-inducing and curvature-sensing proteins and peptides. In this paper, a simple experiment was performed to show qualitatively how membrane remodeling by N-terminal amphipathic helix of Amphiphysin affects the spatial distribution of the transmembrane Fc receptor protein (FcεRI) in mast cells. Results indicate that an elevated concentration of amphipathic helices can interfere with the formation of a typical mast cell synapse.