Structurally and biochemically defining divergent actin and its non-canonical regulators in Giardia

项目总结 感染单细胞寄生虫蓝氏贾第鞭毛虫会导致肠道不适，并可能导致严重的 仍未得到治疗的儿童营养不良。贾第虫每年感染3亿人，其中20% 对一线治疗耐药的病例。贾第鞭毛虫附着在宿主的肠道上皮上，有两个 细胞结构：腹盘和腹外侧凸缘。依恋研究近五十年来的发展 集中在吸盘状的腹盘上，而片状的腹外侧翼则保留下来 在生物化学和结构上都没有特征。已发表的工作和我们的初步数据表明， 细胞骨架蛋白肌动蛋白在腹外侧翼中起着中心作用。贾第鞭毛虫的肌动蛋白差异最大 在真核生物中鉴定的序列，缺乏所有规范的、真核细胞中的肌动蛋白结合蛋白，但肌动蛋白 参与贾第鞭毛虫的几个核心细胞过程，包括胞质分裂和细胞形态形成。同舟共济 这表明贾第虫构建基于肌动蛋白的细胞结构的机制与 其他真核生物，贾第鞭毛虫可能包含非规范调节因子和新的肌动蛋白结合蛋白， 调节肌动蛋白的细胞骨架。本提案描述了表征肌动蛋白(目标1)和 来自贾第鞭毛虫的法兰(Aim 2)，使用冷冻电子显微镜和超分辨率成像。这项工作将定义 法兰超微结构的分子基础(目标2)和揭示保守和发散 贾第鞭毛虫中肌动蛋白的组装、动力学和调节机制(目标1)。确定功能属性， 将贾第鞭毛虫的肌动蛋白细胞骨架与其脊椎动物同源物区分开来将为新药提供机会 专门针对寄生虫而不伤害宿主的。这项工作还将产生基本的见解 转化为肌动蛋白的核心属性。 这个跨学科的研究项目是一个特殊的博士后培训机会，利用 这些问题存在于几个科学领域和加州大学可用的资源的结合点 华盛顿纳入了一项有影响力的研究战略和培训计划。PI将与两名教职员工导师共同工作： Justin Kollman博士，冷冻电子显微镜和发散肌动蛋白专家；Alex Paredez博士，领导者 在对贾第虫的研究中，他开发了许多用于研究寄生虫中的肌动蛋白的工具。此外， 少年派在大学和全国各地组建了一个由合作者和导师组成的团队。这所大学 将在负责任的研究、专业技能和教学方面提供广泛的培训 教育学。通过执行研究战略和培训计划，PI将获得技术专长 在低温电子显微镜和超分辨率显微镜以及关键职业技能方面 一个学术研究实验室。总之，这项研究和培训将为PI进行研究做准备 弥合单个蛋白质与寄主和病原体之间的细胞水平相互作用之间的分辨率差距。

PROJECT SUMMARY Infection by the unicellular parasite Giardia lamblia leads to intestinal distress and can cause severe malnutrition in children who remain untreated. Giardia infects over 300 million people annually, with 20% of those cases being resistant to front-line treatment. Giardia attaches to the intestinal epithelium of its hosts with two cellular structures: the ventral disk and the ventrolateral flange. Attachment research over the past fifty years has focused on the suction-cup like ventral disk, whereas the lamellipodium-like ventrolateral flange remains biochemically and structurally uncharacterized. Published work and our preliminary data indicate that the cytoskeletal protein actin plays a central role in the ventrolateral flange. Giardia has the most divergent actin sequence identified in eukaryotes and lacks all of the canonical, eukaryotic actin-binding proteins, yet actin is involved in several core cellular processes in Giardia, including cytokinesis and cell morphogenesis. Together this suggests that the mechanisms by which Giardia builds actin-based cellular structures differ significantly from other eukaryotes and that Giardia likely contains non-canonical regulators and novel actin-binding proteins that modulate the actin cytoskeleton. This proposal describes approaches to characterize both actin (Aim 1) and the flange (Aim 2) from Giardia using cryo-electron microscopy and super-resolution imaging. This work will define the molecular underpinnings of the flange ultrastructure (Aim 2) and reveal the conserved and divergent mechanisms of actin assembly, dynamics, and regulation in Giardia (Aim 1). Identifying functional properties that distinguish the actin cytoskeleton of Giardia from its vertebrate homolog will provide opportunities for new drugs that specifically target the parasite without harming the host. This work will also generate fundamental insights into core actin properties. This interdisciplinary research project is an exceptional postdoctoral training opportunity, leveraging questions that sit at the nexus of several scientific fields and the resources available at the University of Washington into an impactful research strategy and training plan. The PI will work jointly with two faculty mentors: Dr. Justin Kollman, an expert in cryo-electron microscopy and divergent actins; and Dr. Alex Paredez, a leader in research on Giardia who has developed many of the tools used to study actin in the parasite. In addition, the PI has assembled a team of collaborators and mentors at the University and around the country. The University will provide extensive training in the responsible conduct of research, professional skills, and teaching pedagogies. Though the execution of the research strategy and training plan, the PI will gain technical expertise in cryo-electron microscopy and super-resolution microscopy as well as the key career skills necessary to launch an academic research laboratory. Together, this research and training will prepare the PI to conduct studies bridging the resolution gap between individual proteins and the cell-level interactions of hosts and pathogens.