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

项目摘要 单细胞寄生虫蓝氏贾第鞭毛虫的感染会导致肠道不适，并可能导致严重的 营养不良的儿童仍然没有得到治疗。贾第虫每年感染3亿多人，其中20%的人 对一线治疗产生抗药性的病例。贾第虫附着在宿主的肠上皮上， 细胞结构：腹侧盘和腹外侧凸缘。依恋研究五十年 已经集中在吸盘状腹盘，而板状腹外侧凸缘仍然存在 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凸缘中起中心作用。贾第虫具有最大分歧性肌动蛋白 序列在真核生物中鉴定，缺乏所有典型的真核肌动蛋白结合蛋白，但肌动蛋白是 参与贾第虫的几个核心细胞过程，包括胞质分裂和细胞形态发生。一起 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，贾第虫可能含有非典型的调节剂和新的肌动蛋白结合蛋白， 调节肌动蛋白细胞骨架。该建议描述了表征肌动蛋白（目标1）和 法兰（目标2）从贾第虫使用冷冻电子显微镜和超分辨率成像。这项工作将定义 凸缘超微结构的分子基础（目的2），并揭示了保守和分歧 贾第虫肌动蛋白装配、动力学和调控机制（目的1）。识别功能特性， 将贾第虫的肌动蛋白细胞骨架与其脊椎动物同系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。这项工作也将产生基本的见解 核心肌动蛋白的特性。 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 问题，坐在几个科学领域的联系和可用的资源在大学 华盛顿转化为一个有影响力的研究战略和培训计划。PI将与两名教师导师共同工作： 博士Justin Kollman，低温电子显微镜和发散肌动蛋白专家; Alex Paredez博士，领导者 他开发了许多用于研究寄生虫肌动蛋白的工具。此外该 PI在大学和全国各地组建了一个合作者和导师团队。大学 将提供广泛的培训，负责进行研究，专业技能和教学 我的错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的职业技能， 学术研究实验室这项研究和培训将共同为PI开展研究做好准备 弥合单个蛋白质与宿主和病原体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.