Neutrophils have long been regarded as essential for host defense against Staphylococcus aureus infection. However, survival of the pathogen inside various cells, including phagocytes, has been proposed as a mechanism for persistence of this microorganism in certain infections. Therefore, we investigated whether survival of the pathogen inside polymorphonuclear neutrophils (PMN) contributes to the pathogenesis of S. aureus infection. Our data demonstrate that PMN isolated from the site of infection contain viable intracellular organisms and that these infected PMN are sufficient to establish infection in a naive animal. In addition, we show that limiting, but not ablating, PMN migration into the site of infection enhances host defense and that repletion of PMN, as well as promoting PMN influx by CXC chemokine administration, leads to decreased survival of the mice and an increased bacterial burden. Moreover, a global regulator mutant of S. aureus (sar−) that lacks the expression of several virulence factors is less able to survive and/or avoid clearance in the presence of PMN. These data suggest that the ability of S. aureus to exploit the inflammatory response of the host by surviving inside PMN is a virulence mechanism for this pathogen and that modulation of the inflammatory response is sufficient to significantly alter morbidity and mortality induced by S. aureus infection.

The extent to which ligand occupancy and dimerization contribute to erbB1 signaling is controversial. To examine this, we utilized two-color Quantum Dot tracking for visualization of erbB1 homodimerization and quantification of the dimer off rate (koff) on living cells. Kinetic parameters were extracted using a 3-state Hidden Markov Model to identify transition rates between free, co-confined, and dimerized states. We report that dimers composed of 2 ligand-bound receptors are long-lived and their koff is independent of kinase activity. By comparison, unliganded dimers have >4-fold faster koff. Transient co-confinement of receptors promotes repeated encounters and enhances dimer formation. Mobility decreases >6-fold when ligand-bound receptors dimerize. Blockade of erbB1 kinase activity or disruption of actin networks results in faster diffusion of receptor dimers. These results implicate both signal propagation and the cortical cytoskeleton in reduced mobility of signaling-competent erbB1 dimers.

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 ...

Although much evidence suggests that the plasma membrane of eukaryotic cells is not homogenous, the precise architecture of this important structure has not been clear. Here we use transmission electron microscopy of plasma membrane sheets and specific probes to show that most or all plasma membrane-associated proteins are clustered in cholesterol-enriched domains (''islands'') that are separated by ''protein-free'' and cholesterol-low membrane. These islands are further divided into subregions, as shown by the localization of ''raft'' and ''non-raft'' markers to specific areas. Abundant actin staining and inhibitor studies show that these structures are connected to the cytoskeleton and at least partially depend on it for their formation and/or maintenance.cholesterol ͉ electron microscopy ͉ microdomains ͉ plasma membrane structure

Summary of Recent AdvancesMast cells play as the major effector cells in immediate hypersensitivity through activation via the high-affinity IgE receptor, FcεRI, although many other functions have recently been discovered for this cell type. Given the broad array of proinflammatory mediators secreted from FcεRI-activated mast cells, as well as sensitization to allergens, IgE elevation, and increased mast cells in a majority of atopic dermatitis patients, mast cells are believed to be involved in the pathogenesis of atopic dermatitis. Numerous animal models have been used to study this epidemic disease. Here we review the recent progress to synthesize our current understanding of this disease and potential mechanisms for a mast cell's role in the disease.

We have determined the membrane topography of the high-affinity IgE receptor, FcεRI, and its associated tyrosine kinases, Lyn and Syk, by immunogold labeling and transmission electron microscopic (TEM) analysis of membrane sheets prepared from RBL-2H3 mast cells. The method of Sanan and Anderson (Sanan, D.A., and R.G.W. Anderson. 1991. J. Histochem. Cytochem. 39:1017–1024) was modified to generate membrane sheets from the dorsal surface of RBL-2H3 cells. Signaling molecules were localized on the cytoplasmic face of these native membranes by immunogold labeling and high-resolution TEM analysis. In unstimulated cells, the majority of gold particles marking both FcεRI and Lyn are distributed as small clusters (2–9 gold particles) that do not associate with clathrin-coated membrane. Approximately 25% of FcεRI clusters contain Lyn. In contrast, there is essentially no FcεRI-Syk colocalization in resting cells. 2 min after FcεRI cross-linking, ∼10% of Lyn colocalizes with small and medium-sized FcεRI clusters (up to 20 gold particles), whereas ∼16% of Lyn is found in distinctive strings and clusters at the periphery of large receptor clusters (20–100 gold particles) that form on characteristically osmiophilic membrane patches. While Lyn is excluded, Syk is dramatically recruited into these larger aggregates. The clathrin-coated pits that internalize cross-linked receptors bud from membrane adjacent to the Syk-containing receptor complexes. The sequential association of FcεRI with Lyn, Syk, and coated pits in topographically distinct membrane domains implicates membrane segregation in the regulation of FcεRI signaling.

Engagement of the Fcɛ receptor I (FcɛRI) on mast cells and basophils initiates signaling pathways leading to degranulation. Early activation events include tyrosine phosphorylation of two transmembrane adaptor proteins, linker for activation of T cells (LAT) and non–T cell activation linker (NTAL; also called LAB; a product of Wbscr5 gene). Previous studies showed that the secretory response was partially inhibited in bone marrow–derived mast cells (BMMCs) from LAT-deficient mice. To clarify the role of NTAL in mast cell degranulation, we compared FcɛRI-mediated signaling events in BMMCs from NTAL-deficient and wild-type mice. Although NTAL is structurally similar to LAT, antigen-mediated degranulation responses were unexpectedly increased in NTAL-deficient mast cells. The earliest event affected was enhanced tyrosine phosphorylation of LAT in antigen-activated cells. This was accompanied by enhanced tyrosine phosphorylation and enzymatic activity of phospholipase C γ1 and phospholipase C γ2, resulting in elevated levels of inositol 1,4,5-trisphosphate and free intracellular Ca2+. NTAL-deficient BMMCs also exhibited an enhanced activity of phosphatidylinositol 3-OH kinase and Src homology 2 domain–containing protein tyrosine phosphatase-2. Although both LAT and NTAL are considered to be localized in membrane rafts, immunogold electron microscopy on isolated membrane sheets demonstrated their independent clustering. The combined data show that NTAL is functionally and topographically different from LAT.

Lipid rafts isolated by detergent extraction and sucrose gradient fractionation from mast cells are enriched for the glycosylphosphatidylinositol-linked protein Thy-1, the ganglioside GM1, palmitoylated LAT, and cross-linked IgE receptors, Fc⑀RI. This study addresses the relationship of fractionation data to the organization of raft markers in native membranes. Immunogold labeling and electron microscopy shows there is little or no colocalization of the raft markers Thy-1, GM1, and LAT with each other or with Fc⑀RI on native membrane sheets prepared from unstimulated cells. External cross-linking of Thy-1 promotes coclustering of Thy-1 with LAT, but not with GM1. Thy-1 and LAT clusters occur on membrane regions without distinctive features. In contrast, external cross-linking of Fc⑀RI and GM1 causes their redistribution to electron-dense membrane patches independently of each other and of Thy-1. The distinctive patches that accumulate cross-linked Fc⑀RI and GM1 also accumulate osmium, a stain for unsaturated lipids, and are sites for coated vesicle budding. Electron microscopy reveals a more complex and dynamic topographical organization of membrane microdomains than is predicted by biochemical analysis of detergent-resistant membranes. INTRODUCTIONOrdered regions of membrane, known as microdomains, lipid rafts, detergent-resistant membranes (DRMs), and other abbreviations are thought to be critical sites of signal propagation and membrane trafficking (Edidin, 1997(Edidin, , 2001Simons and Ikonen, 1997;Anderson, 1998; London, 1998, 2000;Jacobson and Dietrich, 1999;Langlet et al., 2000;Anderson and Jacobson, 2002). Analysis of these regions typically begins with detergent solubilization of whole cells followed by sucrose density gradient centrifugation and the recovery of detergent-resistant membranes from the light fractions of the gradient. The DRMs are enriched for caveolin, glycosylphosphatidylinositol-linked (GPI-linked) proteins, glycosphingolipids, GM1 ganglioside, and cholesterol, suggesting that these components are associated in the liquid-ordered (l o ) phase of the lipid bilayer (Schroeder et al., 1994;Ahmed et al., 1997). The interpretation of gradient centrifugation experiments remains controversial. There is evidence that detergents may force associations between components that are not colocalized in intact cells (Mayor and Maxfield, 1995), and fractionation results are known to be dramatically altered by varying the concentration of Triton X-100 (Field et al., 1999;Parolini et al., 1999), by use of alternative detergents (Montixi et al., 1998;Surviladze et al., 1998) or by omission of detergent altogether (Ilangumaran et al., 1999;Harder and Kuhn, 2000;Surviladze et al., 2001). Methods to observe membrane segregation in situ have also generated controversy. Results based on light and fluorescence microscopy of proteins and lipids, including refinements of the established fluorescence recovery after photobleaching methods and new single particle tracking methods, have led some investigators to co...