How do nucleation promoting factors build different actin networks?

成核促进因子如何构建不同的肌动蛋白网络？

基本信息

批准号：
9761279
负责人：
Rachel Marie Brunetti
金额：
$ 3.7万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9761279
关键词：
Actins Affect Atherosclerosis Biological Process Blood Circulation Cells Chronic Clustered Regularly Interspaced Short Palindromic Repeats Communication Complex Cystic Fibrosis Data Defect Disease Endocytic Vesicle Endocytosis Endocytosis Inhibition Ensure Family Family member Functional disorder Generations Genetic Geometry Heart Diseases Hematopoietic Homologous Gene Human Immune Immune response In Vitro Infection Inflammation Inflammatory Interruption Knock-out Lead Leukocytes Light Lung diseases Mediating Membrane Modeling Molecular Movement Neutrophil Infiltration Pathway interactions Pattern Phenotype Play Polymers Process Protein Family Proteins Reperfusion Injury Research Role Signal Pathway Signal Transduction Site Structure System Testing Therapeutic Intervention Tissues Uncertainty Vascular Diseases Work cell motility follow-up in vivo insight interest lung injury migration mutant nanopattern nanoscale neutrophil polymerization preference recruit spatiotemporal

项目摘要

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.

抽象的中性粒细胞穿过身体迁移到感染部位以启动免疫反应。在此期间过程肌动蛋白被组织成影响细胞迁移的板状足和内吞网络。大部分都是这样的肌动蛋白结构出现在成核水平。虽然我们已经确定了多种成核促进剂因子（NPF）及其上游调节器，我们不知道这些NPF如何在空间上排列来构建他们不同的肌动蛋白网络。我对 WASP 家族蛋白特别感兴趣，这是一类控制通过激活 Arp2/3 复合体形成分支肌动蛋白网络。尽管遵循类似途径，该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，最终适合不同的生物功能。例如，WASP 家族成员 WAVE 在前缘形成宽广的传播波，形成扁平肌动蛋白网络底层突出，片状片状伪足。同时，WASP 和 N-WASP 形成点状结构，垂直于膜聚合肌动蛋白以帮助内吞囊泡的分裂。目前，我们做不了解这些 NPF 如何构建不同的结构，无论是在其独特的空间组织还是在其肌动蛋白网络的几何形状。了解 WASP 家族 NPF 在分子水平上的组织结构至关重要，因为它们的失调损害我们发起免疫反应的能力并导致多种病理生理学。具体来说，慢性炎症性疾病，如动脉粥样硬化和阻塞性肺病，是由中性粒细胞增加免疫细胞募集。此外，当这些细胞过度积累时，组织损伤发生，如缺血再灌注损伤和囊性纤维化中的肺损伤。我的研究将测试不同的模型来了解 WASP 家族 NPF 的独特空间组织如何维持。在目标 1 中，我将确定膜几何形状如何在局部发挥作用以招募不同的 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。一旦我有找到并打破了曲率传感机制，我将测试曲率偏好是否单独有效负责 NPF 的空间组织以及随后所需的肌动蛋白聚合的调节定向迁移。然后，在目标 2 中，我将建立 WASP、WAVE 和 N-WASP 各自扮演的角色迁移并确定它们是否相互依赖以进行正确的定位。直到最近，WAVE 仍是这些 NPF 中只有一个被认为参与了前沿的形成。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 基因敲除会导致前沿的 WAVE 定位丢失，表明这些 NPF 之间可能存在通信。我将使用每个 NPF 的 CRISPR 介导的敲除系来研究是否观察到迁移缺陷是由于前缘形成或内吞作用被破坏或 NPF 之间的通讯失调所致。