How do nucleation promoting factors build different actin networks?

摘要 中性粒细胞穿过身体迁移到感染部位以启动免疫应答。在此 过程肌动蛋白被组织成影响细胞迁移的片状伪足和内吞网络。这在很大程度 肌动蛋白结构在成核水平上出现。虽然我们已经确定了各种成核促进 因子（NPF）及其上游调节因子，我们不知道这些NPF在空间上是如何排列的， 不同的肌动蛋白网络。我对WASP家族蛋白特别感兴趣，这是一类NPF， 通过激活Arp 2/3复合物形成分支肌动蛋白网络。尽管类似的 在肌动蛋白通路中，这个家族的成员具有不同的空间组织，导致肌动蛋白的形成。 具有不同几何形状的网络，最终适用于不同的生物功能。例如，WASP 家族成员WAVE在前缘形成宽的传播波，形成扁平肌动蛋白网络 下面有肿胀的片状片状伪足。同时，WASP和N-WASP形成点状结构， 在细胞膜上垂直的肌动蛋白，以帮助内吞囊泡的分裂。目前， 我不明白这些NPF如何建立不同的结构，无论是在其独特的空间组织和在 从而形成肌动蛋白网络的几何结构。 了解WASP家族NPF在分子水平上的组织是至关重要的，因为它们的失调 损害了我们产生免疫反应的能力，并导致多种病理生理学。具体地说， 慢性炎性疾病如动脉粥样硬化和阻塞性肺病是由 增加中性粒细胞的免疫细胞募集。此外，当这些细胞过度积累时， 如在局部缺血-再灌注损伤和囊性纤维化的肺损伤中所见，发生损伤。 我的研究将测试不同的模型，以了解WASP家族NPF的独特空间组织方式 树立政治意识在目标1中，我将确定膜几何结构如何在局部起作用以招募不同的WASP家族 成员我将通过在具有不同曲率的纳米图案基底上电镀细胞来测试这一点。一旦我有 发现，并打破，曲率感应机制，我将测试是否曲率偏好单独 负责NPF的空间组织和随后的肌动蛋白聚合的调节， 定向迁移。然后，在目标2中，我将确定WASP、WAVE和N-WASP各自在以下方面发挥的作用： 迁移并确定它们是否相互依赖以进行适当的本地化。直到最近，WAVE还是 这些NPF中只有一个被认为与前缘形成有关。然而，最近的数据显示， WASP对于正确的迁移也是必要的。此外，我观察到WASP敲除导致 在前缘失去WAVE定位，表明这些NPF之间可能存在通信。我 将对每个NPF使用CRISPR介导的敲除系，以研究观察到的迁移缺陷是否 是由于前缘形成或内吞作用被破坏或NPF之间的通讯失调。

ABSTRACT Neutrophils migrate across the body to sites of infection to initiate an immune response. During this process actin is organized into lamellipodial and endocytic networks that affect cell migration. Much of this actin structure comes in at the level of nucleation. While we have identified a variety of nucleation promoting factors (NPFs) and their upstream regulators, we do not know how these NPFs are spatially arranged to build their different actin networks. I am particularly interested in WASP family proteins, a class of NPFs that control the formation of branched actin networks through activation of the Arp2/3 complex. Despite following similar pathways, members of this family have a different spatial organization that lead to the formation of actin networks with different geometries, ultimately suited for different biological functions. For example, the WASP family member WAVE forms broad, propagating waves at the leading edge that pattern the flat actin network underlying protrusive, sheet-like lamellipodia. Meanwhile, WASP and N-WASP form punctate structures that polymerize actin perpendicular to the membrane to aide in the scission of endocytic vesicles. Presently, we do not understand how these NPFs build different structures, both in their distinct spatial organization and in the resulting geometry of their actin networks. Understanding what organizes WASP family NPFs on a molecular level is crucial, as their disregulation compromises our ability to mount an immune response and results in multiple pathophysiologies. Specifically, chronic inflammatory disorders such as atherosclerosis and obstructive pulmonary diseases arise from to increased immune cell recruitment by neutrophils. Additionally, when these cells hyper-accumulate, tissue damage occurs, as is seen in ischemia-reperfusion injuries and lung damage in cystic fibrosis. My research will test different models for how the distinct spatial organization of WASP family NPFs is maintained. In Aim 1, I will determine how membrane geometry may act locally to recruit different WASP family members. I will test this by plating cells on nanopatterned substrates with varying curvature. Once I have found, and broken, the curvature-sensing mechanism, I will test whether curvature preference alone is responsible for the spatial organization of NPFs and subsequent modulation of actin polymerization needed for directed migration. Then, in Aim 2 I will establish the role that WASP, WAVE and N-WASP each play in migration and determine if they depend on one another for proper localization. Until recently, WAVE was the only one of these NPFs thought to be involved in leading edge formation. However, recent data shows that WASP is also necessary for proper migration. Additionally, I have observed that WASP knockout results in a loss of WAVE localization at the leading edge, suggesting there may be communication between these NPFs. I will use CRISPR-mediated knockout lines for each NPF to investigate whether an observed defect in migration is due to disrupted leading edge formation or endocytosis or from misregulated communication between NPFs.