Temporally resolved interactions between antigen-stimulated IgE receptors and Lyn kinase on living cells

Upon cross-linking by antigen, the high affinity receptor for immunoglobulin E (IgE), FcɛRI, is phosphorylated by the Src family tyrosine kinase Lyn to initiate mast cell signaling, leading to degranulation. Using fluorescence correlation spectroscopy (FCS), we observe stimulation-dependent associations between fluorescently labeled IgE-FcɛRI and Lyn-EGFP on individual cells. We also simultaneously measure temporal variations in the lateral diffusion of these proteins. Antigen-stimulated interactions between these proteins detected subsequent to the initiation of receptor phosphorylation exhibit time-dependent changes, suggesting multiple associations between FcɛRI and Lyn-EGFP. During this period, we also observe a persistent decrease in Lyn-EGFP lateral diffusion that is dependent on Src family kinase activity. These stimulated interactions are not observed between FcɛRI and a chimeric EGFP that contains only the membrane-targeting sequence from Lyn. Our results reveal real-time interactions between Lyn and cross-linked FcɛRI implicated in downstream signaling events. They demonstrate the capacity of FCS cross-correlation analysis to investigate the mechanism of signaling-dependent protein–protein interactions in intact, living cells

Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting

We present an analytical method to quantify clustering in super-resolution localization images of static surfaces in two dimensions. The method also describes how over-counting of labeled molecules contributes to apparent self-clustering and how the effective lateral resolution of an image can be determined. This treatment applies to clustering of proteins and lipids in membranes, where there is significant interest in using super-resolution localization techniques to probe membrane heterogeneity. When images are quantified using pair correlation functions, the magnitude of apparent clustering due to over-counting will vary inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. Over-counting does not yield apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (Fc{\epsilon}RI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM) and scanning electron microscopy (SEM). We find that apparent clustering of labeled IgE bound to Fc{\epsilon}RI detected with both methods arises from over-counting of individual complexes. Thus our results indicate that these receptors are randomly distributed within the resolution and sensitivity limits of these experiments.Comment: 22 pages, 5 figure

The ROM / UWO Mummy Project: A Microcosm of Progress in Mummy Research

The beginnings of the Royal Ontario Museum can be traced back to the excavations and collections of Charles Trick Currelly, a staff member of the Egyptian Exploration Fund in the early 1900s. Currelly excavated with Sir Flinders Petrie at Abydos and with Edouard Naville at Deir el Bahari. With the assistance of Robert Mond and others, Currelly amassed a rich and diverse collection that became the basis for the ROM, which opened its doors in 1914. Part of that collection included several Egyptian mummies (Currelly 1971) . The Egyptologicalholdings at the ROM include eight mummies: one dating to the Predynastic Period, five from the Pharaonic Period, one from the Roman Period and one without context. Two of these, Nakht and Djedmaatesankh, have been well studied by Peter Lewin and associates, while three more are the subjects of the current investigation. The objectives of this poster are to review the work and accomplishments of the previous research, to describe the preliminary results of the current research project and to outline directions for future work

The Ubiquitous Dermokine Delta Activates Rab5 Function in the Early Endocytic Pathway

The expression of the recently identified dermokine (Dmkn) gene leads to four families of proteins with as yet unknown functions. The secreted α, β and γ isoforms share an epidermis-restricted expression pattern, whereas the δ isoform is intracellular and ubiquitous. To get an insight into Dmknδ function, we performed yeast two-hybrid screening and identified the small GTPases Rab5 as partners for Dmknδ. The Rab5 proteins are known to regulate membrane docking and fusion in the early endocytic pathway. GST pull-down assays confirmed the direct interaction between Rab5 and Dmknδ. Transient expression of Dmknδ in HeLa cells led to the formation of punctate structures colocalized with endogenous Rab5 and clathrin, indicating Dmknδ involvement in the early steps of endocytosis. Dmknδ indeed colocalized with transferrin at early stages of endocytosis, but did not modulate its endocytosis or recycling kinetics. We also showed that Dmknδ was able to bind both inactive (GDP-bound) and active (GTP-bound) forms of Rab5 in vitro but preferentially targeted GDP-bound form in HeLa cells. Interestingly, Dmknδ expression rescued the Rab5S34N-mediated inhibition of endosome fusion. Moreover, Dmknδ caused the enlargement of vesicles positive for Rab5 by promoting GTP loading onto the small GTPase. Together our data reveal that Dmknδ activates Rab5 function and thus is involved in the early endosomal trafficking

α1A-Adrenergic Receptor Induces Activation of Extracellular Signal-Regulated Kinase 1/2 through Endocytic Pathway

G protein-coupled receptors (GPCRs) activate mitogen-activated protein kinases through a number of distinct pathways in cells. Increasing evidence has suggested that endosomal signaling has an important role in receptor signal transduction. Here we investigated the involvement of endocytosis in α1A-adrenergic receptor (α1A-AR)-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2). Agonist-mediated endocytic traffic of α1A-AR was assessed by real-time imaging of living, stably transfected human embryonic kidney 293A cells (HEK-293A). α1A-AR was internalized dynamically in cells with agonist stimulation, and actin filaments regulated the initial trafficking of α1A-AR. α1A-AR-induced activation of ERK1/2 but not p38 MAPK was sensitive to disruption of endocytosis, as demonstrated by 4°C chilling, dynamin mutation and treatment with cytochalasin D (actin depolymerizing agent). Activation of protein kinase C (PKC) and C-Raf by α1A-AR was not affected by 4°C chilling or cytochalasin D treatment. U73122 (a phospholipase C [PLC] inhibitor) and Ro 31–8220 (a PKC inhibitor) inhibited α1B-AR- but not α1A-AR-induced ERK1/2 activation. These data suggest that the endocytic pathway is involved in α1A-AR-induced ERK1/2 activation, which is independent of Gq/PLC/PKC signaling

Link between Intestinal CD36 Ligand Binding and Satiety Induced by a High Protein Diet in Mice

CD36 is a ubiquitous membrane glycoprotein that binds long-chain fatty acids. The presence of a functional CD36 is required for the induction of satiety by a lipid load and its role as a lipid receptor driving cellular signal has recently been demonstrated. Our project aimed to further explore the role of intestinal CD36 in the regulation of food intake. Duodenal infusions of vehicle or sulfo-N-succinimidyl-oleate (SSO) was performed prior to acute infusions of saline or Intralipid (IL) in mice. Infusion of minute quantities of IL induced a decrease in food intake (FI) compared to saline. Infusion of SSO had the same effect but no additive inhibitory effect was observed in presence of IL. No IL- or SSO-mediated satiety occurred in CD36-null mice. To determine whether the CD36-mediated hypophagic effect of lipids was maintained in animals fed a satietogen diet, mice were subjected to a High-Protein diet (HPD). Concomitantly with the satiety effect, a rise in intestinal CD36 gene expression was observed. No satiety effect occurred in CD36-null mice. HPD-fed WT mice showed a diminished FI compared to control mice, after saline duodenal infusion. But there was no further decrease after lipid infusion. The lipid-induced decrease in FI observed on control mice was accompanied by a rise in jejunal oleylethanolamide (OEA). Its level was higher in HPD-fed mice than in controls after saline infusion and was not changed by lipids. Overall, we demonstrate that lipid binding to intestinal CD36 is sufficient to produce a satiety effect. Moreover, it could participate in the satiety effect induced by HPD. Intestine can modulate FI by several mechanisms including an increase in OEA production and CD36 gene expression. Furthermore, intestine of mice adapted to HPD have a diminished capacity to modulate their food intake in response to dietary lipids

Segregation of Fluorescent Membrane Lipids into Distinct Micrometric Domains: Evidence for Phase Compartmentation of Natural Lipids?

Background: We recently reported that sphingomyelin (SM) analogs substituted on the alkyl chain by various fluorophores (e.g. BODIPY) readily inserted at trace levels into the plasma membrane of living erythrocytes or CHO cells and spontaneously concentrated into micrometric domains. Despite sharing the same fluorescent ceramide backbone, BODIPY-SM domains segregated from similar domains labelled by BODIPY-D-e-lactosylceramide (D-e-LacCer) and depended on endogenous SM. Methodology/Principal Findings. We show here that BODIPY-SM further differed from BODIPY-D-e-LacCer or -glucosylceramide (GlcCer) domains in temperature dependence, propensity to excimer formation, association with a glycosylphosphatidylinositol (GPI)-anchored fluorescent protein reporter, and lateral diffusion by FRAP, thus demonstrating different lipid phases and boundaries. Whereas BODIPY-D-e-LacCer behaved like BODIPY-GlcCer, its artificial stereoisomer, BODIPY-L-t-LacCer, behaved like BODIPY- and NBD-phosphatidylcholine (PC). Surprisingly, these two PC analogs also formed micrometric patches yet preferably at low temperature, did not show excimer, never associated with the GPI reporter and showed major restriction to lateral diffusion when photobleached in large fields. This functional comparison supported a three-phase micrometric compartmentation, of decreasing order: BODIPY-GSLs > -SM > -PC (or artificial L-t-LacCer). Co-existence of three segregated compartments was further supported by double labelling experiments and was confirmed by additive occupancy, up to ~70% cell surface coverage. Specific alterations of BODIPY-analogs domains by manipulation of corresponding endogenous sphingolipids suggested that distinct fluorescent lipid partition might reflect differential intrinsic propensity of endogenous membrane lipids to form large assemblies. Conclusions/Significance. We conclude that fluorescent membrane lipids spontaneously concentrate into distinct micrometric assemblies. We hypothesize that these might reflect preexisting compartmentation of endogenous PM lipids into non-overlapping domains of differential order: GSLs > SM > PC, resulting into differential self-adhesion of the two former, with exclusion of the latter