A class of highly fluorescent and photostable core−shell nanoparticles from a modified Stöber synthesis in the size range 20−30 nm is described. These are monodisperse solution, 20 times brighter, more than their constituent fluorophore, amenable to specific labeling biological macromolecules for bioimaging experiments. The photophysical characteristics encapsulated fluorophores differ solution properties. This raises possibility tuning nanoparticle structure toward enhanced radiative...

The membrane raft hypothesis postulates the existence of lipid bilayer heterogeneities, or domains, supposed to be important for cellular function, including lateral sorting, signaling, and trafficking. Characterization heterogeneities in live cells has been challenging part because inhomogeneity not usually definable by optical microscopy. Model systems, giant unilamellar vesicles, allow fluorescence discrimination coexisting phase types, but thus far have focused on optically resolvable...

Protein lateral mobility in cell membranes is generally measured using fluorescence photobleaching recovery (FPR). Since the development of this technique, data have been interpreted by assuming free Brownian diffusion surface receptors two dimensions, an interpretation that requires a subset diffusing species remains immobile. The origin so-called immobile fraction mystery. In FPR, motions thousands particles are inherently averaged, inevitably masking details individual motions. Recently,...

We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy of static surfaces two dimensions. use this how over-counting labeled molecules contributes apparent self-clustering calculate the effective lateral resolution image. This treatment applies distributions proteins lipids cell membranes, where there is significant interest techniques probe membrane heterogeneity. When are quantified...

The earliest known step in the activation of high affinity IgE receptor, FcεRI, is tyrosine phosphorylation its β and γ subunits by Src family kinase, Lyn. We report here that aggregation-dependent association FcεRI with specialized regions plasma membrane precedes appears necessary for this event. Tyrosine occurs intact cells only associate these detergent-resistant domains, which are enriched active Furthermore, efficient vitro those associated isolated domains. This highly sensitive to...

Tyrosine phosphorylation of the high affinity immunoglobulin (Ig)E receptor (FcεRI) by Src family kinase Lyn is first known biochemical step that occurs during activation mast cells and basophils after cross-linking FcεRI antigen. The hypothesis specialized regions in plasma membrane, enriched sphingolipids cholesterol, facilitate coupling was tested investigating functional structural effects cholesterol depletion on Lyn/FcεRI interactions. We find with methyl-β-cyclodextrin substantially...

The mechanisms by which a cell uses and adapts its functional membrane organization are poorly understood the subject of ongoing investigation discussion. Here, we study one proposed mechanism: crosslinking components. In immune signaling (and other membrane-associated processes), small change in clustering specific proteins can lead to large-scale reorganizations that involve numerous We have investigated physical effect minor component, ganglioside GM 1 , simple lipid models plasma...

Detergent-resistant plasma membrane structures, such as caveolae, have been implicated in signalling, transport, and vesicle trafficking functions. Using sucrose gradient ultracentrifugation, we isolated low-density, Triton X-100-insoluble domains from RBL-2H3 mucosal mast cells that contain several markers common to including a src-family tyrosine kinase, p53/56lyn. Aggregation of Fc epsilon RI, the high-affinity IgE receptor, causes significant increase amount p53/56lyn associated with...

We recently showed that ligand-mediated cross-linking of FcεRI, the high-affinity receptor for immunoglobulin E, on RBL-2H3 mast cells results in its co-isolation with detergent-resistant membranes (DRM) and consequent tyrosine phosphorylation by co-localized kinase Lyn is a critical early event signaling this [Field et al. (1997) J. Biol. Chem. 272, 4276−4280]. As part efforts to determine structural bases these interactions, we examined phospholipid composition DRM vesicles isolated from...

A fluorescent chemical sensor based upon a core/shell architecture of silica nanoparticles (see figure) is introduced. By covalent attachment organic reference dyes within the core and in outer shell, optimal architectures can be realized. This paves way for development highly functional particles with catalytic or synthetic abilities. Supporting information this article available on WWW under http://www.wiley-vch.de/contents/jc_2296/2006/z600017_s.pdf from author. Please note: The publisher...

ABSTRACT Previous studies showed that crosslinking of IgE-FcεRI complexes on RBL-2H3 mast cells causes their association with isolated detergent-resistant membranes, also known as lipid rafts, in a cholesterol-dependent process precedes initiation signaling by these receptors. To investigate interactions intact cells, we examined the co-redistribution raft components crosslinked using confocal microscopy. After several hours at 4°C, glycosylphosphatidylinositol-linked protein Thy-1 and...

Protein patterning was carried out using a simple procedure based on photolithography wherein the protein not subjected to UV irradiation and high temperatures or contacted with denaturing solvents strongly acidic basic solutions. Self-assembled monolayers of poly(ethylene glycol) (PEG) silicon surfaces were exposed oxygen plasma through patterned photoresist. The etched regions back-filled an initiator for surface-initiated atom transfer radical polymerization (ATRP). ATRP sodium acrylate...

Activation of store operated Ca(2+) entry involves stromal interaction molecule 1 (STIM1), localized to the endoplasmic reticulum (ER), and calcium channel subunit (Orai1/CRACM1), plasma membrane. Confocal microscopy shows that thapsigargin-mediated depletion ER stores in RBL mast cells causes a redistribution STIM1, labeled with monomeric red fluorescent protein (mRFP), micrometer-scale ER-plasma membrane junctions contain Orai1/CRACM1, Aequorea coerulescens green (AcGFP). Using...

Mesoporous silica with cubic symmetry has attracted interest from researchers for some time. Here, we present the room temperature synthesis of mesoporous nanoparticles possessing Pm3̅n very high molar ratios (>50%) 3-aminopropyl triethoxysilane. The is robust allowing, example, co-condensation organic dyes without loss structure. By means pore expander molecules, size can be enlarged 2.7 to 5 nm, while particle decreases. Adding and co-condensing fluorescent in same reduces average further...

The plasma membrane contains ordered lipid domains, commonly called rafts, enriched in cholesterol, sphingolipids, and certain signaling proteins. Lipid rafts play a structural role signal initiation by the high affinity receptor for IgE. Cross-linking of IgE-receptor complexes antigen causes their coalescence with where they are phosphorylated Src family tyrosine kinase, Lyn. To understand how participate functional coupling between Lyn FcϵRI, we investigated whether raft environment...

Specialized plasma membrane domains known as lipid rafts participate in signal transduction and other cellular processes, their liquid ordered (L(o)) phase appears to be important for function. To quantify lipids biological membranes, we investigated steady-state fluorescence anisotropy of two probes, 2-[3-(diphenylhexatrienyl)propanoyl]-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). We...

Strong steric interactions among proteins on crowded living cell surfaces were revealed by measurements of the equilibrium spatial distributions in applied potential gradients. The fraction accessible surface occupied mobile can be accurately represented including exclusion statistical thermodynamic analysis data. analyses enhanced, concentration-dependent activity coefficients, implying unanticipated even at typical receptor concentrations.

Large scale aggregation of fluorescein-labeled immunoglobulin E (IgE) receptor complexes on the surface RBL cells results in co-aggregation a large fraction lipophilic fluorescent probe 3,3'-dihexadecylindocarbocyanine (diI) that labels plasma membranes much more uniformly absence aggregation. Most diI molecules are localized patches aggregated receptors have lost their lateral mobility as determined by fluorescence photobleaching recovery. The outside is mobile, and its similar to control...

The nitroxide-mediated polymerization of styrenic monomers containing oligo(ethylene glycol) (OEGn) moieties was chosen for the preparation biocompatible polymer brushes tethered to silicon oxide surfaces due broad range monomer structures available and use a nonmetallic initiator. These were characterized by near-edge X-ray absorption fine structure water contact angle measurements. biocompatibility these grown studied compared with deposited assemblies surface-bound OEGn-terminated silanes...