The actin cytoskeleton has been implicated in restricting diffusion of plasma membrane components. Here, simultaneous observations of quantum dot-labelled FcεRI motion and GFPtagged actin dynamics provide direct evidence that actin filament bundles define micron-sized domains that confine mobile receptors. Dynamic reorganisation of actin structures occurs over seconds, making the location and dimensions of actin-defined domains time dependent. Multiple FcεRI often maintain extended close proximity without detectable correlated motion, suggesting that they are co-confined within membrane domains. FcεRI signalling is activated by cross-linking with multivalent antigen. We show that receptors become immobilised within seconds of crosslinking. Disruption of the actin cytoskeleton results in delayed immobilisation kinetics and increased diffusion of cross-linked clusters. These results implicate actin in membrane partitioning that not only restricts diffusion of membrane proteins, but also dynamically influences their longrange mobility, sequestration, and response to ligand binding.Signal transduction from the external environment to the cell interior is typically mediated by ligand-bound transmembrane receptors embedded in a lipid bilayer. In many systems, receptor activation is associated with changes in receptor dynamics and membrane topography1 -3 . Among these are the multi-chain immune recognition receptor family members that include the B-cell receptor (BCR) of B-cells, the T-cell receptor (TCR) of Tcells, and the high affinity IgE receptor (FcεRI) of mast cells and basophils, which are crucial to the execution of key events in the immune response. Cross-linking of these transmembrane receptors induces receptor oligomerisation, protein and lipid kinase activation and Ca 2+ mobilisation, leading in turn to cytoskeletal reorganisation, receptor trafficking and cell-specific responses including altered gene expression [4][5][6] . These signalling events have been well studied by biochemical techniques, but the precise mechanism by which oligomerisation initiates these events has remained elusive. Full understanding of these complex signalling cascades will require a more complete description of receptor movements in the membrane, including restrictions that might limit receptor diffusion and accessibility.Correspondence should be addressed to D.S.L. (dlidke@salud.unm.edu). 4 These authors contributed equally to this work. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2011 January 18. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptA rich literature on single particle tracking (SPT) methods to follow the lateral diffusion of transmembrane and membrane-associated proteins7 -10 has revealed nanometer-scale "confinement zones" that restrict lateral diffusion and supports the general notion that plasma membrane organisation is more structured than originally postulated by the fluid mosaic model11. A membrane-skeleton fence (picket fence) model ...

SUMMARY Crosslinking of IgE-bound FcεRI triggers mast cell degranulation. Previous FRAP and phosphorescent anisotropy studies suggested that FcεRI must immobilize to signal. Here, single quantum dot (QD) tracking and hyperspectral microscopy methods are used to redefine relationships between receptor mobility and signaling. QD-IgE-FcεRI aggregates of at least three receptors remain highly mobile over extended times at low concentrations of antigen that induce Syk kinase activation and near-maximal secretion. Multivalent antigen, presented as DNP-QD, also remains mobile at low doses that support secretion. FcεRI immobilization is marked at intermediate and high antigen concentrations, correlating with increases in cluster size and rates of receptor internalization. The kinase inhibitor PP2 blocks secretion without affecting immobilization or internalization. We propose that immobility is a feature of highly crosslinked immunoreceptor aggregates, is a trigger for receptor internalization, and is not required for tyrosine kinase activation leading to secretion.

Surfactant-passivated germanium nanocrystals (Ge(0) NCs) 3-5 nm in diameter were synthesized and encapsulated with functionalized phospholipids to yield water-soluble Ge(0) NCs. Upon encapsulation, the NCs retained their cubic crystalline phase and displayed good resistance to oxidation, as determined by transmission electron microscopy and X-ray photoelectron spectroscopy. As a test of their cell compatibility, the ability of carboxyfluorescein (CF)-labeled dinitrophenyl (DNP)-functionalized Ge(0) NCs to crosslink dinitrophenol-specific immunoglobulin E antibodies on the surface of mast cells (RBL-2H3) was examined in vitro. Treatment with a multivalent DNP antigen (i.e., DNP-Ge(0) NCs or CF-DNP-Ge(0) NCs) caused crosslinking of FcepsilonRI receptors and cellular responses, which were evaluated with morphological and colorimetric assays and live-cell fluorescence microscopy. Incubation of RBL-2H3 cells with Ge(0) NCs for approximately 24 h gave less than a 2 % increase in cell death as compared to DNP-functionalized bovine serum albumin. When irradiated with near-infrared (NIR) radiation (lambda(exc)=770 nm, 1.1 W cm(-2)) from a continuous-wave Ti:sapphire laser, the bulk-solution temperature of a toluene solution containing 20 mg mL(-1) Ge(0) NCs increased by approximately 35 degrees C within 5 min. Phospholipid-encapsulated water-soluble Ge(0) NCs at concentrations of 1.0 mg mL(-1) also displayed stable photothermal behavior under repetitive and prolonged NIR laser exposures in water, to yield a temperature increase of approximately 20 degrees C within 5 min (lambda(exc)=770 nm, 0.9 W cm(-2)). The photothermal efficiency of water-soluble Ge(0) NCs compares favorably with a recent report for Au nanoshells.