No abstract

IQGAP1 is a multi-domain protein that acts as a scaffold for multiple signaling pathways. IQGAP1 generates the lipid messenger PI(3,4,5)P 3 by scaffolding the phosphoinositide kinases PIPKIs and PI3K.The dynamics of this scaffolding protein complex in intact, living cells are unknown. Here, we delineate the role of IQGAP1 in PI(3,4,5)P 3 -mediated signaling in live cells under basal and stimulated conditions using fluorescence lifetime imaging microscopy. We demonstrate that IQGAP1 interacts strongly with PIPKIγ at intracellular entities and on the plasma membrane, and scaffolds PI3K and PIPKIγ in response to physiological changes. Additionally, we show that IQGAP1 scaffolds phosphoinositides with PI3K, PIPKIγ and EGFR, and forms clusters upon cell stimulation with epidermal growth factor. Importantly, we show that IQGAP1 connects PI(3,4,5)P 3 -mediated signaling and cytoskeletal signaling pathways by binding PIPKIγ in proximity of the cytoskeletal proteins talin and Cdc42. Our results support a model in which IQGAP1 mediates crosstalk between phosphoinositide signaling and the cytoskeleton to promote directed cell movement. IQGAP1 connects PI signaling to cytoskeletal reorganization

IntroductionSecretory IgA (SIgA) protects the intestinal epithelium from enteric pathogens such as Salmonella enterica serovar Typhimurium (STm) through a process known as immune exclusion, where invading bacteria are aggregated via antibody cross-linking, encased in mucus, and then cleared from the intestinal tract via peristalsis. At high cell densities, the STm aggregates form a tightly packed network that is reminiscent of early bacterial biofilms. However, the underlying mechanism of how SIgA mediates this transition from a motile and invasive state to an avirulent sessile state in STm is currently unknown.MethodsIn this report, we developed and validated a methodology known as the “snow globe” assay to enable real-time imaging and quantification of STm agglutination by the mouse monoclonal IgA Sal4.ResultsWe observed that agglutination in the snow globe assay was dose-dependent, antigen-specific, and influenced by antibody isotype. We determined that flagellar-based motility was a prerequisite for rapid onset of agglutination, even at high cell densities where cell-cell contacts are expected to be frequent. We also investigated the roles of individual cyclic-di-GMP metabolizing enzymes previously implicated in motility and biofilm formation in Sal4 IgA-mediated agglutination.DiscussionTaken together, our results demonstrate that IgA-mediated agglutination is a dynamic process influenced by bacterial motility and cell-cell collisions. We conclude that the snow globe assay is a viable platform to further decipher the molecular and genetic determinants that drive this interaction.