UVRAG and the VPS34 complex II regulate macropinocytosis via the synthesis of PI(3)P on the plasma membrane 2259463

Abstract Introduction Macropinocytosis is central to critical macrophage immune functions including wound healing, antigen presentation, and resolution of inflammation. Macropinocytosis also facilitates nutrient uptake and growth in macrophages, T cells, and cancer cells. Methods We performed whole-genome screens to identify key regulators of macropinocytosis. Phospho-proteomic analysis was conducted after depletion of the key negative regulator PAK2. Lattice lightsheet microscopy (LLSM), utilizing macrophages expressing 2xFYVE-mScarlet reporter, tracked PI(3)P dynamics in macropinocytic cups and nascent macropinosomes. Results Uvrag and other members of VPS34 complex II, which catalyzes PI(3)P synthesis, was identified as a top positive regulator. Conversely, the loss of PAK2 increased macropinosome formation, confirming it as a key negative regulator. PAK2 depletion reduced phosphorylation of GAPex-5 (Rab5 GEF) and Rabankyrin-5 (Rab5 effector), both of which were required for efficient macropinocytosis. LLSM confirmed PI(3)P localization in macropinocytic cups prior to closure, as well as on large (0.5-3 μm) nascent macropinosomes and smaller (< 0.5 μm) vesicles moving toward the plasma membrane. This data suggests PI(3)P supports macropinocytosis by recruiting GAPex-5, which activates the Rab5/Rabankyrin5/Rabenosyn5 pathway to facilitate SNARE-mediated fusion preceding macropinocytic cup closure. Conclusion This work elucidates the mechanisms underpinning PI(3)P regulation of macropinocytosis and elucidates the mechanisms of PAK2-mediated negative regulation of Rab5 activity and macropinocytosis. Unlocking mechanisms of macropinocytosis may reveal new therapeutic targets for in vivo study of this process and better understanding of the role of macropinocytosis plays in macrophage cell biology. Funding Source NIH COBRE Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)