Deciphering the mechanism of SHIP1 regulation in human neutrophils

摘要 免疫细胞解释其环境中的化学信号，并做出决定，控制其 命运。例如，人类中性粒细胞经常通过极化和迁移来对信号做出反应 化学物质的来源。对这些细胞功能至关重要的是 细胞表面受体、小GTP酶和合成磷脂酰肌醇的酶 磷酸(PIP)脂类。本研究旨在破译控制沟通的机制 在质膜上这些不同类别的信号分子之间。使用人类 中性粒细胞，我们发现CDC42 GTP酶和一种名为SHIP1的脂质磷酸酶触发了一种 以行波的形式在质膜上传播的分子信号。 这种行为被称为兴奋性，涉及到蛋白质重新招募的反复循环。 膜。本研究的目的是确定SHIP1是如何调节兴奋性的 通过整合来自脂类和膜系留蛋白的信号来构建信号网络。 使用各种体外生物化学技术，包括支持膜技术 和单分子成像，我们将确定脂质成分如何控制SHIP1 膜结合和磷酸酶活性(目标1)。使用调节 细胞内可兴奋的信号网络，我们将重建控制SHIP1的机制 膜募集、释放自身抑制和激活(目标2)。同时，我们将使用 基于CRISPR的基因组编辑、光遗传学和荧光定量活细胞成像 生物传感器阐明PIP脂类和小GTP酶之间的通讯是如何调节的 按SHIP1。使用这些工具，我们将确定SHIP1作为信令网络的角色 支架与脂质磷酸酶(目标3)。总的来说，这项研究将统一膜生物物理学。 和细胞生物学来解释PIP脂类、小GTP酶和SHIP1是如何协同控制 中性粒细胞的兴奋性信号网络和细胞迁移。通过解开白血 细胞感知、解释和响应致病信号我们将填补知识空白 关于这些信号分子如何协调细胞运动和潜在的 可兴奋的网络。这一发现可能会为研究人员开发新的疗法打开大门 它可以用来调节免疫细胞的功能，以对抗感染，炎症， 和癌症。

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.