Signal Integration from Membranes to the Actin Cytoskeleton

我的实验室对理解肌动蛋白细胞骨架的基本机制有着长期的兴趣 调节以及这些机制如何驱动基本过程，如细胞迁移，囊泡运输， 免疫细胞活化和神经元形态发生。我们对解决监管问题特别感兴趣 Wiskott-Aldrich综合征蛋白（WASP）家族蛋白的机制。这些蛋白质在 在连接膜信号Arp 2/3介导的肌动蛋白聚合在整个真核细胞中的作用。葛- 这些蛋白质的遗传突变或失调与人类疾病密切相关，包括各种神经系统疾病， 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家族蛋白的调节机制知之甚少，这对联合国的研究构成了很大的障碍。 了解它们在生物学和人类病理学中的作用，并确定治疗相关疾病的新途径， 放松。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WAVE及其在疾病中的作用。WAVE被整合到一个名为WAVE的大型蛋白质组装体中 调节复合物（WRC），这是必不可少的聚合肌动蛋白在质膜上。我们已经阻止- 研究了WRC是如何被两种不同的小GTP酶Rac 1和Arf激活的，疾病相关的突变是如何发生的， 破坏WRC激活，以及WRC如何与各种配体相互作用，包括神经元受体HPO-30， 氧化还原调节剂p47 phox，和肌动蛋白调节剂Ena/VASP/p47 phox-34。未来五年，我们将继续 通过解决一系列关于WAVE监管的新的重要问题，垂直移动该领域。同时， 我们将开始探索另外两种WASP家族蛋白质WHAMM和JMY的机制， 尽管它们在调节肌动蛋白在内膜的组装中起着重要作用，但其机制却知之甚少。我们 将结合联合收割机蛋白质工程，定量生物化学和质谱，单分子荧光， 荧光显微镜和单粒子低温电子显微镜（cryo-EM），以确定WRC是如何活动的。 通过Arf结合，不同的Arf和Rac 1结合位点如何建立协同性，以及WRC如何相互作用 与几个新的配体重要的神经元形态发生，轴突的指导，和寄生虫感染。此外， 为了解剖单个配体的机制，我们将建立基于膜的单分子荧光- 荧光实验以确定多种配体，包括肌醇磷脂， GTP酶和膜蛋白在非常类似于细胞膜的环境中协同激活WRC。 膜此外，我们将确定如何通过结合各种配体来调节WHAMM和JMY。 我们的工作将提供一个全面的机制框架，了解WASP家族蛋白调节， 是的。这些知识将广泛适用于研究肌动蛋白相关过程的不同领域。合作 与各种生物学家和临床医生，我们的工作将揭示如何突变WASP家族蛋白质来源于 患者的功能中断，并为开发新型干预剂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.