NKG2D is an activation receptor that allows natural killer (NK) cells to detect diseased host cells. The engagement of NKG2D with corresponding ligand results in surface modulation of the receptor and reduced function upon subsequent receptor engagement. However, it is not clear whether in addition to modulation the NKG2D receptor complex and/or its signaling capacity is preserved. We show here that the prolonged encounter with tumor cell-bound, but not soluble, ligand can completely uncouple the NKG2D receptor from the intracellular mobilization of calcium and the exertion of cell-mediated cytolysis. However, cytolytic effector function is intact since NKG2D ligand-exposed NK cells can be activated via the Ly49D receptor. While NKG2D-dependent cytotoxicity is impaired, prolonged ligand exposure results in constitutive interferon ␥ (IFN␥) production, suggesting sustained signal IntroductionNumerous activation receptors can trigger natural killer (NK) cell-mediated cytotoxic and/or cytokine responses. The NKG2D receptor is specific for endogenous ligands (ie, histocompatibility 60 (H60), retinoic acid early inducible gene-1 (RAE-1), and mouse UL16-binding proteinlike transcript 1 (MULT1) in mice), which are usually not expressed on normal resting cells but which are readily induced upon different forms of cellular distress (for a review see Raulet 1 ). Thus, the de novo expression of NKG2D ligands renders cells susceptible to NK cell-mediated lysis. On many tumor cell lines of distinct tissue origins, NKG2D ligands are constitutively expressed. The ectopic expression of NKG2D ligands induces the rejection of several transplantable tumor cell lines. 2,3 Despite NKG2D's prominent role in NK cell activation and T-cell costimulation, it is noteworthy that many established tumor cell lines constitutively express NKG2D ligands, suggesting that tumors can escape NKG2D recognition. Indeed, T and NK cells, which were infiltrating major histocompatibility class I chain-related protein A (MICA)-positive tumors had reduced NKG2D cell surface levels. 4,5 In vitro, soluble MICA or tumor cells expressing MICA induced the down-modulation and degradation of NKG2D expressed by CD8 ϩ T cells, which led to reduced effector functions. 4 In addition, NK cells derived from nonobese diabetic (NOD) mice (a strain of mice prone to autoimmune diabetes) displayed impaired NKG2D function. 6 NKG2D dysfunction and modulation were explained by an interaction of NKG2D with its ligands, which are aberrantly induced on NOD NK cells upon culture. 6 In general, reduced NKG2D function is thought to be the consequence of and directly related to the lower NKG2D cell surface levels. However, it has not been determined whether sustained NKG2D engagement leaves the NKG2D receptor complex and/or its signal transduction capacity intact. To this end we established a culture system in which NK cells are exposed to tumor cells expressing NKG2D ligand. We show that chronic engagement with tumor cell-bound NKG2D ligand can uncouple NKG2D recognition from the ex...

SignificanceThe passive translocation mechanism of arginine-rich cell-penetrating peptides has puzzled the scientific community for more than 20 y. In this study we propose a hitherto unrecognized mechanism of passive cell entry involving fusion of multilamellar structures generated by the cell-penetrating peptides. The geometry of entry for this mechanism is completely different from previously suggested direct translocation mechanisms, leading to another paradigm for designing molecular carriers for drug delivery to the cell.

Lipid membranes can spontaneously organize their components into domains of different sizes and properties. The organization of membrane lipids into nanodomains might potentially play a role in vital functions of cells and organisms. Model membranes represent attractive systems to study lipid nanodomains, which cannot be directly addressed in living cells with the currently available methods. This review summarizes the knowledge on lipid nanodomains in model membranes and exposes how their specific character contrasts with large-scale phase separation. The overview on lipid nanodomains in membranes composed of diverse lipids (e.g., zwitterionic and anionic glycerophospholipids, ceramides, glycosphingolipids) and cholesterol aims to evidence the impact of chemical, electrostatic, and geometric properties of lipids on nanodomain formation. Furthermore, the effects of curvature, asymmetry, and ions on membrane nanodomains are shown to be highly relevant aspects that may also modulate lipid nanodomains in cellular membranes. Potential mechanisms responsible for the formation and dynamics of nanodomains are discussed with support from available theories and computational studies. A brief description of current fluorescence techniques and analytical tools that enabled progress in lipid nanodomain studies is also included. Further directions are proposed to successfully extend this research to cells.

TCR signal strength is involved in many cell fate decisions in the T-cell lineage. Here, we show that transcriptional events induced by Hedgehog (Hh) signaling reduced TCR signal strength in mice. Activation of Hh signaling in thymocytes in vivo by expression of a transgenic transcriptional-activator form of Gli2 (Gli2ΔN2) changed the outcome of TCR ligation at many stages of thymocyte development, allowing self-reactive cells to escape clonal deletion; reducing transgenic TCR-mediated positive selection; reducing the ratio of CD4/CD8 single-positive (SP) cells; and reducing cell surface CD5 expression. In contrast, in the Shh−/− thymus the ratio of CD4/CD8 cells and both positive and negative selection of a transgenic TCR were increased, demonstrating that Shh does indeed influence TCR repertoire selection and the transition from double-positive (DP) to SP cell in a physiological situation. In peripheral T cells, Gli2ΔN2 expression attenuated T-cell activation and proliferation, by a mechanism upstream of ERK phosphorylation.

Ever since technologies enabled the characterization of eukaryotic plasma membranes, heterogeneities in the distributions of its constituents were observed. Over the years this led to the proposal of various models describing the plasma membrane organization such as lipid shells, picket-and-fences, lipid rafts, or protein islands, as addressed in numerous publications and reviews. Instead of emphasizing on one model we in this review give a brief overview over current models and highlight how current experimental work in one or the other way do not support the existence of a single overarching model. Instead, we highlight the vast variety of membrane properties and components, their influences and impacts. We believe that highlighting such controversial discoveries will stimulate unbiased research on plasma membrane organization and functionality, leading to a better understanding of this essential cellular structure.

SummarySignalling molecules integrate, codify and transport information in cells. Organisation of these molecules in complexes and clusters improves the efficiency, fidelity and robustness of cellular signalling. Here, we summarise current views on how signalling molecules assemble into macromolecular complexes and clusters and how they use their physical properties to transduce environmental information into a variety of cellular processes. In addition, we discuss recent innovations in live-cell imaging at the sub-micrometer scale and the challenges of object (particle) tracking, both of which help us to observe signalling complexes and clusters and to examine their dynamic character.

Leukocyte migration to sites of inflammation is a multistep process involving transient adhesion to the endothelium followed by cell surface-controlled proteolysis for transmigration through the vessel wall and chemotactic movement within tissues. One of the key players in this machinery appears to be the urokinase-type plasminogen activator (uPA)/uPA receptor system. The role of uPA and its receptor (CD87) in plasminogen (Plg) activation, cell adhesion, and chemotaxis is well established; however, less is known of how these activities are regulated. Here we provide evidence that the mannose 6-phosphate/insulin-like growth factor 2 receptor (CD222) controls CD87-mediated functions. Expression of human CD222 in CD222؊/؊ mouse fibroblasts downregulated Plg activation, cell adhesion, and chemotaxis induced by the uPA/CD87 system. In addition, we demonstrate that the N-terminal region of CD222, which is similar to the Plg-binding site of streptokinase, plays a crucial role in binding of CD87 and Plg. A peptide derived from this region in CD222 is able to disrupt the physical interaction of CD222 with CD87 and, furthermore, mimics the inhibitory effects of CD222 on CD87 functions. Taken together, our results indicate a novel role for CD222 in regulation of fibrinolysis, cell adhesion, and migration.

Cholesterol-and glycosphingolipid-enriched membrane lipid microdomains, frequently called lipid rafts, are thought to play an important role in the spatial and temporal organisation of immunological synapses. Higher ordering of lipid acyl chains was suggested for these entities and imaging of membrane order in living cells during activation can therefore help to understand the mechanisms responsible for the supramolecular organisation of molecules involved in the activation of T cells. Here, we employ the phase-sensitive membrane dye di-4-ANEPPDHQ together with a variety of spectrally-resolved microscopy techniques, including 2-channel ratiometric TIRF microscopy and fluorescence lifetime imaging, to characterize membrane order at the T cell immunological synapse at high spatial and temporal resolution in live cells at physiological temperature. We find that higher membrane order resides at the immunological synapse periphery where proximal signalling through the immunoreceptors and accessory proteins Corresponding author: Marek Cebecauer, National Heart and Lung Institute, Imperial College, London, UK; m.cebecauer@imperial.ac.uk. NIH Public Access