Patrick W. Yacono

Stress granules and processing bodies are dynamically linked sites of mRNP remodeling

Stress granules (SGs) are cytoplasmic aggregates of stalled translational preinitiation complexes that accumulate during stress. GW bodies/processing bodies (PBs) are distinct cytoplasmic sites of mRNA degradation. In this study, we show that SGs and PBs are spatially, compositionally, and functionally linked. SGs and PBs are induced by stress, but SG assembly requires eIF2α phosphorylation, whereas PB assembly does not. They are also dispersed by inhibitors of translational elongation and share several protein components, including Fas-activated serine/threonine phosphoprotein, XRN1, eIF4E, and tristetraprolin (TTP). In contrast, eIF3, G3BP, eIF4G, and PABP-1 are restricted to SGs, whereas DCP1a and 2 are confined to PBs. SGs and PBs also can harbor the same species of mRNA and physically associate with one another in vivo, an interaction that is promoted by the related mRNA decay factors TTP and BRF1. We propose that mRNA released from disassembled polysomes is sorted and remodeled at SGs, from which selected transcripts are delivered to PBs for degradation.

CFTR is a pattern recognition molecule that extracts Pseudomonas aeruginosa LPS from the outer membrane into epithelial cells and activates NF-κB translocation

Immune cells are activated during cellular responses to antigen by two described mechanisms: (i) direct uptake of antigen and (ii) extraction and internalization of membrane components from antigen-presenting cells. Although endocytosis of microbial antigens by pattern recognition molecules (PRM) also activates innate immunity, it is not known whether this involves extraction and internalization of microbial surface components. Epithelial cells on mucosal surfaces use a variety of receptors that are distinct from the classical endocytic PRM to bind and internalize intact microorganisms. Nonclassical receptor molecules theoretically could act as a type of endocytic PRM if these molecules could recognize, bind, extract, and internalize a pathogen-associated molecule and initiate cell signaling. We report here that the interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) and the outer core oligosaccharide of the lipopolysaccharide (LPS) in the outer membrane of Pseudomonas aeruginosa satisfies all of these conditions. P. aeruginosa LPS was specifically recognized and bound by CFTR, extracted from the organisms surface, and endocytosed by epithelial cells, leading to a rapid (5- to 15-min) and dynamic translocation of nuclear transcription factor NF-κB. Inhibition of epithelial cell internalization of P. aeruginosa LPS prevented NF-κB activation. Cellular activation depended on expression of wild-type CFTR, because both cultured ΔF508 CFTR human airway epithelial cells and lung epithelial cells of transgenic-CF mice failed to endocytose LPS and translocate NF-κB. CFTR serves as a critical endocytic PRM in the lung epithelium, coordinating the effective innate immune response to P. aeruginosa infection.

Endothelial Cell Cortactin Coordinates Intercellular Adhesion Molecule-1 Clustering and Actin Cytoskeleton Remodeling during Polymorphonuclear Leukocyte Adhesion and Transmigration

Endothelial cell ICAM-1 interacts with leukocyte β2 integrins to mediate adhesion and transmit outside-in signals that facilitate leukocyte transmigration. ICAM-1 redistribution and clustering appear necessary for leukocyte transmigration, but the mechanisms controlling ICAM-1 redistribution and clustering have not been identified. We recently reported that Src kinase phosphorylation of endothelial cortactin regulates polymorphonuclear cell (PMN) transmigration. In this study, we tested the hypotheses that the Src family kinase-cortactin pathway mediates association of ICAM-1 with the actin cytoskeleton and that this association is required for ICAM-1 clustering and leukocyte transmigration. Cross-linking ICAM-1 induced cytoskeletal remodeling and a decrease in ICAM-1 lateral mobility, as assessed by fluorescence recovery after photobleaching. Cytoskeletal remodeling after ICAM-1 cross-linking was reduced by knockdown of cortactin by small interfering RNA, by expression of a cortactin mutant deficient in Src phosphorylation sites (cortactin3F), and by the Src kinase inhibitor PP2. Pretreatment of cytokine-activated human endothelial monolayers with cortactin small interfering RNA significantly decreased both actin and ICAM-1 clustering around adherent PMN and the formation of actin-ICAM-1 clusters required for PMN transmigration. Our data suggest a model in which tyrosine phosphorylation of cortactin dynamically links ICAM-1 to the actin cytoskeleton, enabling ICAM-1 to form clusters and facilitate leukocyte transmigration.

CD151 restricts the α6 integrin diffusion mode.

Laminin-binding integrins (α3β1, α6β1, α6β4, α7β1) are almost always expressed together with tetraspanin CD151. In every coexpressing cell analyzed to date, CD151 makes a fundamental contribution to integrin-dependent motility, invasion, morphology, adhesion and/or signaling. However, there has been minimal mechanistic insight into how CD151 affects integrin functions. In MDA-MB-231 mammary cells, tetraspanin CD151 knockdown impairs α6 integrin clustering and functions without decreasing α6 integrin expression or activation. Furthermore, CD151 knockdown minimally affects the magnitude of α6 integrin diffusion, as measured using single particle tracking. Instead, CD151 knockdown has a novel and unexpected dysregulating effect on the mode of α6 integrin diffusion. In control cells α6 integrin shows mostly random-confined diffusion (RCD) and some directed motion (DMO). In sharp contrast, in CD151-knockdown cells α6 integrin shows mostly DMO. In control cells α6 diffusion mode is sensitive to actin disruption, talin knockdown and phorbol ester stimulation. By contrast, CD151 knockdown cell α6 integrin is sensitive to actin disruption but desensitized to talin knockdown or phorbol ester stimulation, indicating dysregulation. Both phorbol ester and EGF stimulate cell spreading and promote α6 RCD in control cells. By contrast, CD151-ablated cells retain EGF effects but lose phorbol-ester-stimulated spreading and α6 RCD. For α6 integrins, physical association with CD151 promotes α6 RCD, in support of α6-mediated cable formation and adhesion. By comparison, for integrins not associated with CD151 (e.g. αv integrins), CD151 affects neither diffusion mode nor αv function. Hence, CD151 support of α6 RCD is specific and functionally relevant, and probably underlies diverse CD151 functions in skin, kidney and cancer cells.

Immune interferon gamma inhibits translational mobility of a plasma membrane protein in preimplantation stage mouse embryos: A T-helper 1 mechanism for immunologic reproductive failure ☆ ☆☆ ★ ★★

OBJECTIVE Immune interferon gamma adversely affects mouse embryo development and has been proposed as a mediator of reproductive failure involving T-helper 1 immunity. We hypothesized that one mechanism by which interferon gamma could exert an adverse effect on embryos was by altering plasma membrane organization and transmembrane protein mobility. STUDY DESIGN The fluorescence photobleaching recovery technique was used to measure the effect of the T-helper 1 cytokine interferon gamma on the translational mobility of a specific embryonic surface glycoprotein recognized by the monoclonal antibody S75. RESULTS Compared with controls interferon gamma significantly decreased the fractional mobility of fluorescein isothiocyanate S75 in one- and two-cell mouse embryos. CONCLUSION Interferon gamma may alter plasma membrane domains or cytoskeletal organization in early-stage embryos. By restricting plasma membrane protein mobility interferon gamma could effect T-helper 1-mediated reproductive failure.