Regulation of T Cell Extravasation and Trafficking by the Actin Cytoskeleton

淋巴细胞在组织中的进入和迁移在免疫监视和执行免疫应答中起关键作用。 效应子功能以及在疾病如自身免疫和淋巴细胞源性肿瘤转移中的作用 恶性肿瘤。为了定位于组织内，淋巴细胞外渗穿过血管内皮细胞壁， 跟随组织中存在的环境线索。在组织内外渗和迁移期间， 淋巴细胞改变形状并延伸膜突起，例如丝状伪足。然而， 关于肌动蛋白网络重塑的特定调节器， 及其在T细胞渗出和间质迁移中的特定作用。我们的目标是确定 线性肌动蛋白网络重塑在介导T细胞外渗和间质运动中的作用。大多数工作在 肌动蛋白细胞骨架重塑集中在上游调节因子，如Rho-GTP酶， 对淋巴细胞功能的多效性作用。在这里，我们专注于线性肌动蛋白的下游效应子 网络重塑、Formin和Ena/Vasp蛋白。这些细胞骨架效应物控制着 丝状伪足中发现的线性肌动蛋白丝，我们预计它们将具有更明确的功能，例如使 寻路调节淋巴细胞外渗和间质迁移。 淋巴细胞经历一个迁移寻路过程，以找到允许的外渗部位 沿着血管壁，并在间质迁移过程中遵循环境线索。丝状伪足可以介导 环境刺激的感觉，并已被建议在淋巴细胞跨内皮细胞 迁移（TEM）。然而，线状肌动蛋白聚合和丝状伪足在促进淋巴细胞增殖中的作用， 外渗和间质运动仍不清楚。我们假设在淋巴细胞 外渗和间质运动需要线性肌动蛋白聚合的细胞骨架效应物来产生 特殊的膜突起，如丝状伪足。我们提出了三个目标，以解决这一关键差距， 了解淋巴细胞外渗和间质运动如何受Formin和Ena/Vasp调节 目的1：确定线性肌动蛋白聚合的细胞骨架效应物在调节 在TEM期间寻路。目的2：确定线性肌动蛋白聚合对细胞骨架效应物的要求 在体内T细胞外渗和组织浸润。目的3：确定线性细胞骨架效应物的作用。 肌动蛋白聚合在间质运动和组织定位期间T细胞寻路中的作用。 我们将使用互补的体内和体外方法，包括尖端的活体内2光子 显微镜下观察，以了解形成蛋白和Ena/Vasp细胞骨架效应物如何调节淋巴细胞外渗 和间质运动性。确定肌动蛋白细胞骨架调节运动的分子基础， 淋巴细胞的外渗可以导致设计新的治疗工具来调节淋巴细胞 运输和免疫应答以治疗自身免疫或预防淋巴细胞肿瘤的转移。

Lymphocyte entry and migration in tissues play a critical role in immune surveillance and execution of effector functions as well as in diseases such as autoimmunity and metastasis of lymphocyte-derived malignancies. To localize within tissues lymphocytes extravasate across the vascular endothelial cell wall and follow environmental cues present in the tissues. During extravasation and migration within tissues, lymphocytes change shape and extend membrane protrusions, such as filopodia. However, little is known about the specific regulators of actin network remodeling necessary to generate these membrane protrusions and their specific role in T cell extravasation and interstitial migration. Our goal is to determine the mechanistic role of linear actin network remodeling in mediating T cell extravasation and interstitial motility. Most work on actin cytoskeletal remodeling has focused on upstream regulators, such as Rho-GTPases, which have pleiotropic effects on lymphocyte functions. Here instead, we focus on downstream effectors of linear actin network remodeling, Formin and Ena/Vasp proteins. These cytoskeletal effectors control the polymerization of linear actin filaments found in filopodia and we expect they will have a more defined function, such as enabling pathfinding, in regulating lymphocyte extravasation and interstitial migration. Lymphocytes undergo a migratory pathfinding process in order to find permissive extravasation sites along vascular walls and to follow environmental cues during interstitial migration. Filopodia can mediate sensing of environmental stimuli and have been suggested to play a role in lymphocyte trans-endothelial migration (TEM). However, the function of linear actin polymerization and of filopodia in promoting lymphocyte extravasation and interstitial motility remains unclear. We hypothesize that pathfinding during lymphocyte extravasation and interstitial motility requires cytoskeletal effectors of linear actin polymerization to generate specialized membrane protrusions, such as filopodia. We propose three aims to address this crucial gap in our understanding of how lymphocyte extravasation and interstitial motility are regulated by Formin and Ena/Vasp proteins: Aim 1: Determine the role of cytoskeletal effectors of linear actin polymerization in regulating pathfinding during TEM. Aim 2: Define the requirement for cytoskeletal effectors of linear actin polymerization in T cell extravasation and tissue infiltration in vivo. Aim 3: Determine the role of cytoskeletal effectors of linear actin polymerization in T cell pathfinding during interstitial motility and tissue localization. We will use complementary in vivo and in vitro approaches, including cutting-edge intra-vital 2-photon microscopy, to understand how Formin and Ena/Vasp cytoskeletal effectors regulate lymphocyte extravasation and interstitial motility. Determining the molecular basis by which the actin cytoskeleton regulates motility and extravasation of lymphocytes can lead to the design of new therapeutic tools to modulate lymphocyte trafficking and immune responses to treat autoimmunity or prevent metastasis of lymphocytic tumors.