CRISPR/Cas9 whole genome screen identifies UVRAG and members of VPS34 complex II as regulators of macrophage macropinocytosis 4573

Abstract Description Macropinocytosis is central to macrophage immune functions including wound healing, antigen presentation, and resolution of inflammation. It also facilitates nutrient uptake and growth in a variety of cells. CRISPR/Cas9 whole-genome screens identified genes regulating Lucifer yellow uptake, a marker of macropinocytosis, in murine bone-marrow derived macrophages (BMDM). UVRAG and other members of VPS34 complex II, which catalyze the formation of PI(3)P from phosphatidylinositol, were identified as top positive regulators of macropinocytosis. Targeted disruption of Uvrag and Pik3r3 revealed that VPS34-II is required for efficient macropinocytosis with UvragsgRNA BMDM having fewer, larger macropinosomes that fail to appropriately traffic. In contrast, depletion of ATG14, a unique member of VPS34-I increases macropinocytosis. Lattice lightsheet microscopy of BMDM expressing 2xFYVE-mScarlet showed two classes of PI(3)P vesicles in BMDM: 1) large (0.5-3 μm) nascent macropinosomes that acquire PI(3)P following scission from the plasma membrane and move toward the nucleus, and 2) smaller (<0.5 μm) PI(3)P puncta that appear to move toward the plasma membrane. As the screens identified numerous genes encoding FYVE- and PX-domain containing proteins, we hypothesize that these may be effectors of PI(3)P by facilitating either delivery of secretory vesicles to the plasma membrane to enable membrane fusogenicity and/or by promoting downstream trafficking and maturation of macropinosomes. Funding Sources R15GM139162 and P20GM135008 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)