Project Summary/Abstract Macropinocytosis, or “cell drinking,” is central to several macrophage functions including wound healing, antigen presentation, and resolution of inflammation. However, there are large gaps in the mechanistic understanding of this process. The long-term goal of this project is to identify novel mediators and cellular mechanisms of macropinocytosis. The overall objective is to investigate how macrophages regulate actin dynamics, phosphoinositide signaling and macropinocytotic efficiency in response to pro- and anti- inflammatory stimuli. The central hypothesis is that the balance of SHP1 phosphatase and SYK kinase activity optimizes the macrophage “actin economy” for macropinocytosis or other actin-dependent processes depending on the activation state of the cell. This hypothesis stems from preliminary CRISPR/Cas9 whole genome screen data produced in the applicant's laboratory indicating that SHP1 and SYK are key regulators of macropinocytosis. The hypothesis will be tested by pursuing two specific aims: 1) Determine the effect of SYK/SHP1 balance on actin dynamics and phosphoinositide signaling at forming macropinosomes; and 2) Determine how ITAM and ITIM containing immune receptors modulate macropinocytosis via the SHP1/SYK balance in resting and activated macrophages. Under the first aim, an already proven live-cell imaging approach established by the applicant will be used to image actin and phosphoinositide dynamics in wildtype or CRISPR/Cas9 gene-disrupted primary murine macrophages with resting, inflammatory, or anti-inflammatory activation states. Under the second aim, mechanisms of SHP1 and SYK activation by recruitment to immune receptors will be tested using fluorescent protein chimeras of SHP1 or SYK in wild type or gene-disrupted macrophages in response to inflammatory or anti-inflammatory stimuli. This approach is innovative because the hypothesis was generated from novel mediators of macropinocytosis identified by a CRISPR/Cas9 whole genome screen. Furthermore, this strategy uses targeted gene disruptions in combination with cellular fluorescent probes and advanced live-cell microscopy techniques for three-dimensional live-cell imaging of the spatiotemporal dynamics of actin polymerization and phosphoinositide signaling during macropinocytosis. The proposed research is significant because it is expected to expand the understanding of novel mechanisms macropinocytosis and the role of macropinocytosis in inflammation. Ultimately, such knowledge has the potential to identify therapeutic targets for modulation of macropinocytosis efficiency and the treatment of inflammatory diseases such as chronic wounds or auto-immune disorders.