SummaryThe proto-oncogenic epidermal growth factor receptor (EGFR) is a tyrosine kinase whose sensitivity to growth factors and signal duration determines cellular behavior. We resolve how EGFR's response to epidermal growth factor (EGF) originates from dynamically established recursive interactions with spatially organized protein tyrosine phosphatases (PTPs). Reciprocal genetic PTP perturbations enabled identification of receptor-like PTPRG/J at the plasma membrane and ER-associated PTPN2 as the major EGFR dephosphorylating activities. Imaging spatial-temporal PTP reactivity revealed that vesicular trafficking establishes a spatially distributed negative feedback with PTPN2 that determines signal duration. On the other hand, single-cell dose-response analysis uncovered a reactive oxygen species-mediated toggle switch between autocatalytically activated monomeric EGFR and the tumor suppressor PTPRG that governs EGFR's sensitivity to EGF. Vesicular recycling of monomeric EGFR unifies the interactions with these PTPs on distinct membrane systems, dynamically generating a network architecture that can sense and respond to time-varying growth factor signals.

Although transfer of membrane proteins has been shown to occur during immune cell interactions, the functional significance of this process is not well understood. Here we describe the intercellular transfer of NKG2D and MHC class I chain-related molecule (MIC) B proteins at the cytotoxic natural killer cell immune synapse (cNK-IS). MICB expressed on the 721.221 cell line induced clustering of NKG2D at the central supramolecular activation cluster, surrounded by a peripheral supramolecular activation cluster containing F-actin. Moreover, natural killer (NK) cell membrane-connective structures formed during cytotoxic interactions contained F-actin, perforin, and NKG2D. NKG2D transfer depended on binding to MICB and was specific because transfer of other molecules not involved in NK-IS formation was not observed. Transfer of MICB to NK cells also was noted, suggesting a bidirectional exchange of receptor͞ligand pairs at cNK-IS. Experiments designed to test the functional significance of these observations revealed that brief interactions between NK cells and MICB expressing target cells led to a reduction in NKG2D-dependent NK cytotoxicity. These data demonstrate interchange of an activating receptor and its ligand at the cNK-IS and document a correlation between synapse organization, intercellular protein transfer, and compromised NK cell function after interaction with a susceptible target cell.cytotoxicity ͉ protein transfer ͉ innate immunity ͉ NKG2D N KG2D, a member of the killer cell lectin-like receptor family, is one of the best characterized natural killer (NK) cellactivating receptors, but it also is expressed on TCR␥␦ ϩ and CD8 ϩ TCR␣␤ ϩ T cells, where it can act as a costimulatory molecule for T cell activation. NKG2D associates with DAP10, a transmembrane adaptor molecule containing a YINM motif that binds and activates via phosphatidylinositol 3-kinase and Grb2 (1, 2). In humans, NKG2D-immune activation can be triggered by interaction with its ligands: the polymorphic MHC class I chain-related molecule (MIC) A and MICB and the UL16-binding proteins (3). NKG2D ligands are absent, or expressed only at low levels, on normal cells, but their expression is often enhanced͞induced on tumors and cells infected by various pathogens. Dysregulation of NKG2D ligand expression with inappropriate activation of NKG2D ϩ T cells also has been reported recently to be a feature of various autoimmune diseases (4).Receptor-ligand interactions between cells do not occur randomly, rather the groups of interacting molecules are found in ordered structures, named immune synapses (IS), providing a platform for intercellular communication among cells of the immune system (5). Upon NK cell-target cell interaction, highly organized supramolecular clusters are formed at the contact area of both cells, termed the NK cell IS (NK-IS) (6, 7). When NK cells interact with susceptible target cells, the redistribution of signaling molecules at the central (c) SMAC (supramolecular activation cluster, SMAC), as well as LFA-1 and talin at th...

Extracellular stimuli are transduced inside the cell by posttranslational modifications (PTMs), such as phosphorylation, of proteins in signaling networks. Insight into the structure of these networks requires quantification of PTM levels in individual cells. Fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to image PTM levels in situ. FLIM on cell arrays that express fluorescent protein fusions can quantify tyrosine phosphorylation patterns in large networks in individual cells. We identified tyrosine kinase substrates by imaging their phosphorylation levels after inhibition of protein tyrosine phosphatases. Analysis of the correlation between protein phosphorylation and expression levels at single cell resolution allowed us to identify positive feedback motifs. Using FLIM on cell arrays (CA-FLIM), we uncovered components that transduce signals from epidermal growth factor receptor.

Cell surface lectin receptors play important roles in the function of macrophages. Herein, we have identified and characterized the human orthologue of the mouse Mcl/Clecsf8. Human CLECSF8 codes for a type II membrane glycoprotein of 215 amino acids that belongs to the human calcium-dependent lectin family (C-type lectin). The cytoplasmic tail of CLECSF8 lacks consensus signaling motifs and its extracellular region shows a single carbohydrate recognition domain (CRD). The CLECSF8 gene has been localized on the telomeric region of the NK gene complex on chromosome 12p13 close to MINCLE. CLECSF8 mRNA shows a monocyte/macrophage expression pattern. Biochemical analysis of CLECSF8 on transiently transfected cells showed a glycoprotein of 30 kDa. Cross-linking of the receptor leads to a rapid internalization suggesting that CLECSF8 constitutes and endocytic receptor.

The interaction of the activating receptor NKG2D with its ligands plays an important role in immunosurveillance of tumors and infectious pathogens, but dysregulation of this system may lead to autoimmunity.

Fluorescence lifetime imaging microscopy (FLIM) can be used to quantify molecular reactions in cells by detecting fluorescence resonance energy transfer (FRET). Confocal FLIM systems based on time correlated single photon counting (TCSPC) methods provide high spatial resolution and high sensitivity, but suffer from poor signal to noise ratios (SNR) that complicate quantitative analysis. We extend a global analysis method, originally developed for single frequency domain FLIM data, with a new filtering method optimized for FRET-FLIM data and apply it to TCSPC data. With this approach, the fluorescent lifetimes and relative concentrations of free and interacting molecules can be reliably estimated, even if the SNR is low. The required calibration values of the impulse response function are directly estimated from the data, eliminating the need for reference samples. The proposed method is efficient and robust, and can be routinely applied to analyze FRET-FLIM data acquired in intact cells.

The 2B4 molecule (CD244) has been described as a coreceptor in human NK cell activation. However, the behavior of 2B4 during the cytotoxic NK cell immune synapse (NK-IS) formation remains undetermined. In this study, we demonstrate the redistribution of 2B4 and the signaling adaptor molecule, signaling lymphocyte activation molecule-associated protein (SAP), to the cytotoxic NK-IS upon formation of conjugates between resting NK cells and EBV-infected 721.221 human cells. Confocal microscopy showed that 2B4 localized at the central supramolecular activation cluster, surrounded by a peripheral supramolecular activation cluster containing talin within NK cell and ICAM-1 on target cells. Videomicroscopy studies with 2B4-GFP-transfected NK cells revealed that 2B4 redistributed to cytotoxic NK-IS as soon as the cell contact occurred. Simultaneously, a SAP-GFP also clustered at the contact site, where it remained during the interaction period. The 2B4 molecular clusters remained bound to the target cell even after NK cell detachment. These results underscore the function of 2B4 as an adhesion molecule and suggest a relevant role in the initial binding, scanning of target cells, and formation of cytotoxic NK-IS. Finally, these findings are indicative of an important role of the activating 2B4/signaling lymphocyte activation molecule-associated protein complex during the recognition of EBV-infected cells.

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