Elucidating mechanisms of macropinosome formation, stabilization, and trafficking

About This Grant

TITLE. Elucidating mechanisms of macropinosome formation, stabilization, and trafficking. SUMMARY/ABSTRACT Macropinocytosis, or “cell drinking,” is central to critical macrophage immune functions including wound healing, antigen presentation, and resolution of inflammation. Macropinocytosis is the non-specific and receptor independent internalization of solutes by the formation of large endocytic structures. Macropinocytosis facilitates nutrient uptake and growth in macrophages, T cells, and numerous types of cancer cells. Despite its significance, there are large gaps in the mechanistic understanding of this process. The long-term goals of this project are to identify novel mediators and cellular mechanisms of macropinocytosis. The central hypothesis is that UVRAG in cooperation with VPS34-II provides positive regulation of macropinocytosis by facilitating recruitment of effectors mediating stabilization, maturation and trafficking while PAK2 provides countervailing negative regulation. This hypothesis stems from CRISPR/Cas9 whole genome screen data produced in the applicant’s laboratory indicating that UVRAG, members of VPS34 complex II (VPS34-II), and Rab5 activators and effectors are key positive regulators of macropinocytosis. New phosphoproteome data provides a strong cross-validation of our CRISPR screens as Gapvd1/GAPex5 (Rab5 activator) and Ankfy1/Rabankyrin5 (Rab5 effector) were identified as putative downstream targets of PAK2. Rabankyrin5 and Rabenosyn5 facilitate SNARE-mediated vesicle fusion. Preliminary data confirms that PAK2 negatively regulates bulk solute uptake and could provide an explanation for the long-standing question of why Rac1 GTP-GDP cycling is required for macropinocytosis. The hypothesis will be tested by pursuing three specific aims: 1) Determine how UVRAG and VPS34-II stabilize macropinosomes following formation and facilitate transit through the cortical actin, 2) Determine the roles of GAPex5, Rabankyrin5, and Rabenosyn5 in vesicle fusion events promoting macropinosome sealing, and 3) Determine how PAK2 provides negative regulation of macropinocytosis. Under the first aim, gene editing methods already established in the applicant’s laboratory will be used to create macrophages that are deficient in UVRAG and other members of VPS34-II. Macropinosome formation, PI(3)P production, and actin dynamics will be imaged via live-cell 3D microscopy to observe spatiotemporal organization of PI(3)P and depolymerization of the cortical actin. The second aim will investigate whether PI(3)P supports macropinocytosis by recruiting GAPex-5, a Rab5 guanine exchange factor, leading to the activation of Rab5 and its effectors Rabankyrin5 and Rabenosyn5, proteins that facilitate SNARE-mediated membrane fusion. Pull-down assays will be used compare Rab5 activity and binding partners in wildtype and Gapvd1sgRNA BMDM. The third aim will investigate how kinase activity of PAK2 inhibits sealing of the macropinocytic cup by measuring Lucifer yellow uptake with or without pharmacologic inhibition of PAK2 following photoactivation of Rac1 and identify proteins downstream of PAK2 mediating PAK2 inhibition of macropinocytosis.