Deciphering the mechanism of SHIP1 regulation in human neutrophils

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Abstract Immune cells interpret chemical cues in their environment and make decisions that control their fate. For example, human neutrophils often respond to signals by polarizing and migrating toward the chemical source. Critical for these cellular functions is the dynamic interplay between cell surface receptors, small GTPases, and the enzymes that synthesize phosphatidylinositol phosphate (PIP) lipids. This study aims to decipher the mechanisms the control communication between these different classes of signaling molecules at the plasma membrane. Using human neutrophils, we find that Cdc42 GTPase and a lipid phosphatase denoted SHIP1 trigger a molecular signal that propagates across the plasma membrane in the form of a traveling wave. This behavior, coined excitability, involves repetitive cycles of protein recruitment ON and OFF the membrane. The goal of this study is to determine how SHIP1 regulates the excitable signaling network by integrating signals derived from lipids and membrane tethered proteins. Using a variety of in vitro biochemistry techniques, including supported membrane technology and single molecule imaging, we will determine how lipid composition controls SHIP1 membrane association and phosphatase activity (Aim 1). Using factors that regulate the excitable signaling network in cells, we will reconstitute mechanisms that control SHIP1 membrane recruitment, release of autoinhibition, and activation (Aim 2). In parallel, we will use CRISPR based genome editing, optogenetics, and quantitative live cell imaging of fluorescent biosensors to elucidate how communication between PIP lipids and small GTPase is regulated by SHIP1. Using these tools we will determine the role SHIP1 serves as signaling network scaffold versus a lipid phosphatase (Aim 3). Overall, this study will unify membrane biophysics and cell biology to explain how PIP lipids, small GTPases, and SHIP1 synergistically control the excitable signaling network and cell migration in neutrophils. By unraveling how white blood cells sense, interpret, and respond to pathogenic signals we will fill a gap in knowledge concerning how these signaling molecules coordinate cellular movement with the underlying excitable network. This discovery could open doors for researchers to develop new therapeutics that can be used to modulate immune cell functions in ways that combat infection, inflammation, and cancer.