The interferon regulatory factor family member 8 (IRF8) regulates differentiation of lymphoid and myeloid lineage cells by promoting or suppressing lineage-specific genes. How IRF8 promotes hematopoietic progenitors to commit to one lineage while preventing the development of alternative lineages is not known. Here we report an IRF8-EGFP fusion protein reporter mouse that revealed previously unrecognized patterns of IRF8 expression. Differentiation of hematopoietic stem cells into oligopotent progenitors is associated with progressive increases in IRF8-EGFP expression. However, significant induction of IRF8-EGFP is found in granulocyte-myeloid progenitors (GMPs) and the common lymphoid progenitors (CLPs) but not the megakaryocytic-erythroid progenitors. Surprisingly, IRF8-EGFP identifies three subsets of the seemingly homogeneous GMPs with an intermediate level of expression of EGFP defining bipotent progenitors that differentiation into either EGFPhi monocytic progenitors or EGFPlo granulocytic progenitors. Also surprisingly, IRF8-EGFP revealed a highly heterogeneous pre-pro-B population with a fluorescence intensity ranging from background to 4 orders above background. Interestingly, IRF8-EGFP readily distinguishes true B cell-committed (EGFPint) from those that are non-committed. Moreover, dendritic cell progenitors expressed extremely high levels of IRF8-EGFP. Taken together, the IRF8-EGFP reporter revealed previously unrecognized subsets with distinct developmental potentials in phenotypically well-defined oligopotent progenitors, providing new insights into the dynamic heterogeneity of developing hematopoietic progenitors.

Anthrax toxin action requires triggering of natural endocytic transport mechanisms whereby the binding component of the toxin forms channels (PA63) within endosomal limiting and intraluminal vesicle membranes to deliver the toxin’s enzymatic components into the cytosol. Membrane lipid composition varies at different stages of anthrax toxin internalization, with intraluminal vesicle membranes containing ~70% of anionic bis(mono-acylglycero)phosphate lipid. Using model bilayer measurements, we show that membrane lipids can have a strong effect on the anthrax toxin channel properties, including the channel-forming activity, voltage-gating, conductance, selectivity, and enzymatic factor binding. Interestingly, the highest PA63 insertion rate was observed in bis(monoacylglycero)phosphate membranes. The molecular dynamics simulation data show that the conformational properties of the channel are different in bis(monoacylglycero)phosphate compared to PC, PE, and PS lipids. The anthrax toxin protein/lipid bilayer system can be advanced as a novel robust model to directly investigate lipid influence on membrane protein properties and protein/protein interactions.

Obstructing conductive pathways of the channel-forming toxins with targeted blockers is a promising drug design approach. Nearly all tested positively charged ligands have been shown to reversibly block the cation-selective channel-forming protective antigen (PA 63) component of the binary anthrax toxin. The cationic ligands with more hydrophobic surfaces, particularly those carrying aromatic moieties, inhibited PA 63 more effectively. To understand the physical basis of PA 63 selectivity for a particular ligand, detailed information is required on how the blocker structural elements (e.g., positively charged and aromatic groups) influence the molecular kinetics of the blocker/channel binding reactions. In this study, we address this problem using the highresolution single-channel planar lipid bilayer technique. Several structurally distinct cationic blockers, namely per-6-S-(3-amino) propyl-b-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-a-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-b-cyclodextrin, per-6-S-(3-aminomethyl) benzyl-g-cyclodextrin, methyltriphenylphosphonium ion, and G0 polyamidoamine dendrimer are tested for their ability to inhibit the heptameric and octameric PA 63 variants and PA 63 F427A mutant. The F427 residues form a hydrophobic constriction region inside the channel, known as the ''f-clamp.'' We show that the cationic blockers interact with PA 63 through a combination of forces. Analysis of the binding reaction kinetics suggests the involvement of cation-p, Coulomb, and salt-concentration-independent p-p or hydrophobic interactions in the cationic cyclodextrin binding. It is possible that these blockers bind to the f-clamp and are also stabilized by the Coulomb interactions of their terminal amino groups with the water-exposed negatively charged channel residues. In PA 63 F427A, only the suggested Coulomb component of the cyclodextrin interaction remains. Methyltriphenylphosphonium ion and G0 polyamidoamine dendrimer, despite being positively charged, interact primarily with the f-clamp. We also show that seven-and eightfold symmetric cyclodextrins effectively block the heptameric and octameric forms of PA 63 interchangeably, adding flexibility to the earlier formulated blocker/target symmetry match requirement.