Signal Integration from Membranes to the Actin Cytoskeleton

我的实验室对了解肌动蛋白细胞骨架的基本机制有长期的兴趣。 调控以及这些机制如何推动基本过程，如细胞迁移、囊泡运输、 免疫细胞激活和神经元形态发生。我们对解决这一规定特别感兴趣 Wiskott-Aldrich综合征蛋白(WASP)家族蛋白的机制。这些蛋白质发挥着中枢作用 在真核细胞中连接膜信号与Arp2/3介导的肌动蛋白聚合的作用。GE- 这些蛋白的磁性突变或调控不当与人类疾病密切相关，包括各种神经细胞疾病。 发育障碍、免疫综合征、病原体感染和癌症。尽管它们很重要， 大多数WASP家族蛋白的调控机制知之甚少，这为非WASP家族蛋白的研究提供了很大的障碍。 了解它们在生物学和人类病理学中的作用，寻找治疗相关疾病的新途径。 放松点。我们最近的研究为理解WASP-家族做出了一系列关键贡献 蛋白质波及其在疾病中的作用。WAVE被整合到一个名为WAVE的大型蛋白质组件中 调节复合体(WRC)，它是肌动蛋白在质膜上聚合所必需的。我们已经阻止了- 挖掘WRC是如何被两个不同的小GTP酶--rac1和Arf激活的，疾病相关突变是如何发挥作用的 WRC的激活，以及WRC如何与不同的配体相互作用，包括神经元受体HPO-30， 氧化还原调节子p47Phox和肌动蛋白调节子Ena/Vasp/UNC-34。未来五年，我们将继续 通过解决一系列关于波浪调节的新的重要问题，垂直移动领域。同时， 我们将开始探索另外两个WASP家族蛋白WHAMM和JMY的机制，对于这两个蛋白，非常 尽管它们在调节内膜的肌动蛋白组装中起着重要的作用，但在机制上却知之甚少。我们 将结合蛋白质工程，定量生物化学和质谱学，单分子荧光- Cence显微镜和单粒子低温电子显微镜(Cryo-EM)来确定WRC是如何发挥作用的。 受Arf结合的影响，不同的Arf和rac1结合位点如何建立协作性，以及WRC如何相互作用 有几个新的配体对神经元形态发生、轴突引导和寄生虫感染很重要。另外-- 针对单个配体的解剖机制，我们将建立基于膜的单分子荧光。 通过实验来确定包括肌醇磷脂在内的多个配体， GTP酶和膜蛋白在一种与细胞膜密切相似的环境中协同激活WRC- 薄膜。此外，我们将确定WHAMM和JMY是如何通过与各种配体结合来调节的。 我们的工作将为理解WASP家族蛋白调控提供一个全面的机制框架。 提顿。这些知识将对研究肌动蛋白相关过程的不同领域有广泛的帮助。在协作中 与不同的生物学家和临床医生一起，我们的工作将揭示WASP家族蛋白的突变是如何从 患者对功能的干扰为新型干预剂的开发提供了新的思路和靶点。

My laboratory has a long-term interest in understanding the basic mechanisms underlying actin cytoskeleton regulation and how these mechanisms drive fundamental processes such as cell migration, vesicle trafficking, immune cell activation, and neuron morphogenesis. We are particularly interested in solving the regulation mechanisms of the Wiskott-Aldrich Syndrome Protein (WASP) family proteins. These proteins play a central role in linking membrane signals to the Arp2/3-mediated actin polymerization throughout eukaryotic cells. Ge- netic mutation or misregulation of these proteins is heavily involved in human diseases, including various neu- rodevelopmental disorders, immune syndromes, pathogen infections, and cancer. Despite their importance, the regulation mechanisms of most WASP-family proteins are poorly understood, posing a large barrier to un- derstanding their roles in biology and human pathology and identifying new avenues of treating related dis- eases. Our recent studies have made a series of pivotal contributions to the understanding of the WASP-family protein WAVE and its role in disease. WAVE is incorporated in a large protein assembly named the WAVE Regulatory Complex (WRC), which is essential to polymerizing actin at the plasma membrane. We have deter- mined how WRC is activated by two distinct small GTPases, Rac1 and Arf, how disease-related mutations dis- rupt WRC activation, and how WRC interacts with various ligands, including the neuronal receptor HPO-30, the redox regulator p47phox, and the actin regulator Ena/VASP/UNC-34. In the next five years, we will continue to move the field vertically by addressing a series of new, important questions about WAVE regulation. In parallel, we will start exploring the mechanisms of two other WASP-family proteins, WHAMM and JMY, for which very little is known mechanistically despite their essential roles in regulating actin assembly at endomembranes. We will combine protein engineering, quantitative biochemistry and mass spectrometry, single molecule fluores- cence microscopy, and single particle cryogenic electron microscopy (cryo-EM) to determine how WRC is acti- vated by Arf binding, how distinct Arf and Rac1 binding sites establish cooperativity, and how WRC interacts with several novel ligands important to neuron morphogenesis, axon guidance, and parasite infection. In addi- tion to dissecting mechanisms of individual ligands, we will establish membrane-based single-molecule fluores- cence experiments to determine the mechanisms by which multiple ligands, including inositol phospholipids, GTPases, and membrane proteins, cooperatively activate the WRC in a context closely resembling cell mem- branes. Furthermore, we will determine how WHAMM and JMY are regulated by binding to various ligands. Our work will provide a comprehensive mechanistic framework for understanding WASP family protein regula- tion. This knowledge will be broadly useful to different fields studying actin-related processes. In collaboration with various biologists and clinicians, our work will reveal how mutations in WASP-family proteins derived from patients disrupt function and provide new ideas and targets for the development of novel intervening agents.