Continuous engagement of a self-specific activation receptor induces NK cell tolerance

Natural killer (NK) cell tolerance mechanisms are incompletely understood. One possibility is that they possess self-specific activation receptors that result in hyporesponsiveness unless modulated by self–major histocompatability complex (MHC)–specific inhibitory receptors. As putative self-specific activation receptors have not been well characterized, we studied a transgenic C57BL/6 mouse that ubiquitously expresses m157 (m157-Tg), which is the murine cytomegalovirus (MCMV)–encoded ligand for the Ly49H NK cell activation receptor. The transgenic mice were more susceptible to MCMV infection and were unable to reject m157-Tg bone marrow, suggesting defects in Ly49H+ NK cells. There was a reversible hyporesponsiveness of Ly49H+ NK cells that extended to Ly49H-independent stimuli. Continuous Ly49H–m157 interaction was necessary for the functional defects. Interestingly, functional defects occurred when mature wild-type NK cells were adoptively transferred to m157-Tg mice, suggesting that mature NK cells may acquire hyporesponsiveness. Importantly, NK cell tolerance caused by Ly49H–m157 interaction was similar in NK cells regardless of expression of Ly49C, an inhibitory receptor specific for a self-MHC allele in C57BL/6 mice. Thus, engagement of self-specific activation receptors in vivo induces an NK cell tolerance effect that is not affected by self-MHC–specific inhibitory receptors

The Second Transmembrane Domain of P2X7 Contributes to Dilated Pore Formation

Activation of the purinergic receptor P2X7 leads to the cellular permeability of low molecular weight cations. To determine which domains of P2X7 are necessary for this permeability, we exchanged either the C-terminus or portions of the second transmembrane domain (TM2) with those in P2X1 or P2X4. Replacement of the C-terminus of P2X7 with either P2X1 or P2X4 prevented surface expression of the chimeric receptor. Similarly, chimeric P2X7 containing TM2 from P2X1 or P2X4 had reduced surface expression and no permeability to cationic dyes. Exchanging the N-terminal 10 residues or C-terminal 14 residues of the P2X7 TM2 with the corresponding region of P2X1 TM2 partially restored surface expression and limited pore permeability. To further probe TM2 structure, we replaced single residues in P2X7 TM2 with those in P2X1 or P2X4. We identified multiple substitutions that drastically changed pore permeability without altering surface expression. Three substitutions (Q332P, Y336T, and Y343L) individually reduced pore formation as indicated by decreased dye uptake and also reduced membrane blebbing in response to ATP exposure. Three others substitutions, V335T, S342G, and S342A each enhanced dye uptake, membrane blebbing and cell death. Our results demonstrate a critical role for the TM2 domain of P2X7 in receptor function, and provide a structural basis for differences between purinergic receptors. © 2013 Sun et al

Differential Requirements for Clathrin-dependent Endocytosis at Sites of Cell–Substrate Adhesion

Little is known about the influences of cell–substrate attachment in clathrin-mediated endocytosis. We find that cell–substrate adhesion reduces the rate of endocytosis. In addition, we demonstrate that actin assembly is differentially required for efficient endocytosis, with a stronger requirement for actin dynamics at sites of adhesion

Distinct Dynamics of Endocytic Clathrin-Coated Pits and Coated Plaques

Here we classify endocytic structures at the adherent (bottom) surface of many cells in culture into shorter-lived coated pits and longer-lived coated plaques which internalize by different mechanisms

Buses, cars, bicycles and walkers the influence of the type of human transport on the flight responses of waterbirds

One way to manage disturbance to waterbirds in natural areas where humans require access is to promote the occurrence of stimuli for which birds tolerate closer approaches, and so cause fewer responses. We conducted 730 experimental approaches to 39 species of waterbird, using five stimulus types (single walker, three walkers, bicycle, car and bus) selected to mimic different human management options available for a controlled access, Ramsar-listed wetland. Across species, where differences existed (56% of 25 cases), motor vehicles always evoked shorter flight-initiation distances (FID) than humans on foot. The influence of stimulus type on FID varied across four species for which enough data were available for complete cross-stimulus analysis. All four varied FID in relation to stimuli, differing in 4 to 7 of 10 possible comparisons. Where differences occurred, the effect size was generally modest, suggesting that managing stimulus type (e.g. by requiring people to use vehicles) may have species-specific, modest benefits, at least for the waterbirds we studied. However, different stimulus types have different capacities to reduce the frequency of disturbance (i.e. by carrying more people) and vary in their capacity to travel around important habita

Virus Movements on the Plasma Membrane Support Infection and Transmission between Cells

How viruses are transmitted across the mucosal epithelia of the respiratory, digestive, or excretory tracts, and how they spread from cell to cell and cause systemic infections, is incompletely understood. Recent advances from single virus tracking experiments have revealed conserved patterns of virus movements on the plasma membrane, including diffusive motions, drifting motions depending on retrograde flow of actin filaments or actin tail formation by polymerization, and confinement to submicrometer areas. Here, we discuss how viruses take advantage of cellular mechanisms that normally drive the movements of proteins and lipids on the cell surface. A concept emerges where short periods of fast diffusive motions allow viruses to rapidly move over several micrometers. Coupling to actin flow supports directional transport of virus particles during entry and cell-cell transmission, and local confinement coincides with either nonproductive stalling or infectious endocytic uptake. These conserved features of virus–host interactions upstream of infectious entry offer new perspectives for anti-viral interference

Albumin and mammalian cell culture: implications for biotechnology applications

Albumin has a long historical involvement in design of media for the successful culture of mammalian cells, in both the research and commercial fields. The potential application of albumins, bovine or human serum albumin, for cell culture is a by-product of the physico-chemical, biochemical and cell-specific properties of the molecule. In this review an analysis of these features of albumin leads to a consideration of the extracellular and intracellular actions of the molecule, and importantly the role of its interactions with numerous ligands or bioactive factors that influence the growth of cells in culture: these include hormones, growth factors, lipids, amino acids, metal ions, reactive oxygen and nitrogen species to name a few. The interaction of albumin with the cell in relation to these co-factors has a potential impact on metabolic and biosynthetic activity, cell proliferation and survival. Application of this knowledge to improve the performance in manufacturing biotechnology and in the emerging uses of cell culture for tissue engineering and stem cell derived therapies is an important prospect