Defining cytoskeletal mechanisms driving cell motility in Naegleria

定义耐格里虫细胞驱动细胞运动的细胞骨架机制

• 批准号: 10510010
• 负责人: Katrina Velle
• 金额: $ 9.71万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-25 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10510010
• 关键词: Actins; Address; Advisory Committees; Amoeba genus; Animals; Architecture; Automobile Driving; Behavior; Biochemistry; Biological; Biological Assay; Biological Models; Biology; Brain; Bulla; Cell physiology; Cells; Cellular biology; Complement; Complex; Cytoskeleton; Defect; Dictyostelium discoideum; Disease; Drug Targeting; Eating; Electron Microscopy; Encephalitis; Ensure; Environment; Eukaryota; Foundations; Goals; Gold; Health; Human; In Vitro; Individual; Infection; Laboratories; Leukocytes; Measures; Membrane; Meningitis; Mentors; Mentorship; Methods; Microfilaments; Microscopy; Molecular; Morphology; Naegleria; Naegleria fowleri; Organism; Parasites; Pathogenesis; Pathogenicity; Pathway interactions; Phase; Phylogenetic Analysis; Physical environment; Plants; Platinum; Property; Proteins; Pyrenes; Regulation; Research; Research Personnel; Signal Transduction; Speed; Surface; Techniques; Testing; Thinness; Total Internal Reflection Fluorescent; Training; WASP protein; Work; Yeasts; base; brain tissue; career; cell behavior; cell motility; constriction; cranium; defined contribution; directional cell; experimental study; insight; knock-down; light microscopy; member; migration; nanoscale; pathogen; programs; recruit; success

PROJECT SUMMARY/ABSTRACT Although actin is highly conserved throughout eukarya, the mechanisms used to regulate its assembly and disassembly vary across phyla. Precisely timed and placed actin assembly orchestrates nearly every cellular process, including cell migration. While actin-driven cell migration has been defined in some detail in animal cells, it is unknown if diverse eukaryotic pathogens operate using the same set of rules. This proposal will address the hypothesis that there are conserved principles of cell migration by investigating Naegleria, which diverged from the “yeast-to-human” lineage over a billion years ago. Specifically, this work will define the contributions of the cytoskeleton to cell crawling in the “brain-eating amoeba” Naegleria fowleri: a pathogen that crawls into and within the brain, causing a deadly form of meningitis for which there are no reliable treatments. Dr. Velle’s initial postdoctoral research using the commonly-used, non-pathogenic model system Naegleria gruberi highlights the potential for universal rules of motility; N. gruberi crawls on flat surfaces using thin, actin- based protrusions assembled by proteins called the Arp2/3 complex. This actin and Arp2/3 based mechanism is also how animal cells crawl on flat surfaces. However, outside of laboratory conditions, cells rarely—if ever—crawl on flat, uniform surfaces. Animal cells are well-known to switch to a different mode of motility when crawling through complex, restrictive environments, but this has not been tested in Naegleria. Because N. fowleri crawl through narrow channels in the skull and into the brain, despite no known chemotactic signals, dissecting cell migration in restrictive environments is essential for understanding disease. Therefore, Aim 1 will determine mechanisms of cell crawling under confinement at the level of cell behavior. Aim 2 will focus on the actin networks in cells; while the protrusions driving N. gruberi migration on flat surfaces look similar to those of animal cells, defining the actin architecture using Platinum Replica Electron Microscopy (PREM) will reveal if the ultrastructure is conserved. This aim will also provide critical training to complement Dr. Velle’s background in light microscopy. The world expert in PREM, Dr. Svitkina, will provide this training as a member of the scientific advisory committee. Aim 3 will use biochemistry—a technique the applicant has no prior training in—to examine the upstream mechanisms of Arp2/3 complex activation. Dr. Velle has recruited Dr. Bruce Goode, an expert actin biochemist, for this training. Because the leading labs in the field of cell migration frequently employ both microscopy and in vitro actin biochemistry, the proposed training in PREM and biochemistry will ensure the applicant is skilled in the techniques required for success. Dr. Velle has also recruited Dr. Matt Welch, an actin expert, Dr. Meg Titus, who has expertise in actin and amoebae, and Dr. Jim Morris, an expert in N. fowleri, to her scientific advisory committee to provide scientific and career mentoring. Collectively, the proposed work will provide the technical training, and career mentorship required to launch Dr. Velle’s career as an independent investigator with a research program focused on Naegleria’s migration.

项目摘要/摘要 尽管肌动蛋白在整个Eukarya中都是高度保守的，但用于调节其组装的机制和 拆卸在门中各不相同。精确定时并放置肌动蛋白组件几乎每个蜂窝 过程，包括细胞迁移。尽管肌动蛋白驱动的细胞迁移已在动物中详细定义 细胞，未知潜水员真核病原体是否使用相同的规则运作。该提议将 解决以下假设，即通过研究纳格勒（Naegleria）有保守的细胞迁移原则 在十亿年前，与“酵母到人类”的血统有所不同。具体来说，这项工作将定义 细胞骨架对“脑食大脑变形虫” Naegleria Fowleri中细胞爬网的贡献：一种病原体 这会爬到大脑内外，导致脑膜炎的致命形式，没有可靠的 治疗。 Velle博士使用常用的非致病模型系统的最初的博士后研究 Naegleria Gruberi强调了普遍的运动规则的潜力。 N. Gruberi使用 薄的基于肌动蛋白的蛋白质由称为ARP2/3复合物的蛋白质组装。此肌动蛋白和ARP2/3 机制也是动物细胞在平坦表面上爬行的方式。但是，在实验室条件之外，细胞 如果永远，很少在平坦，均匀的表面上爬行。动物细胞是众所周知的，可以切换到不同的模式 在爬行复杂，限制性环境时的运动性，但在纳格勒里亚（Naegleria）尚未进行测试。 因为N. Fowleri爬过头骨的狭窄通道并进入大脑 趋化信号，在限制性环境中剖析细胞迁移对于理解疾病至关重要。 因此，AIM 1将在细胞行为水平下确定细胞爬行的机制。 AIM 2将重点放在细胞中的肌动蛋白网络上；而驱动N. Gruberi迁移在平坦表面上的突起 看起来与动物细胞相似，使用铂副本电子显微镜定义肌动蛋白结构 （PERM）将揭示是否保存超微结构。这个目标还将为完成提供关键的培训 Velle博士的光学显微镜背景。 PREM的世界专家Svitkina博士将提供此培训 科学咨询委员会成员。 AIM 3将使用生物化学 - 申请人没有的技术 先前训练ARP2/3复合激活的上游机制。 Velle博士已招募 本培训专家Bruce Goode博士。因为细胞领域的领先实验室 迁移经常同时采用显微镜和体外肌动蛋白生物化学，这是PREM的拟议培训 并且生物化学将确保申请人熟练获得成功所需的技术。 Velle博士也有 招募了Actin专家Matt Welch博士Meg Titus博士，他在Actin和Amoebae中拥有专业知识，以及Jim博士 莫里斯（N. 拟议的工作总的来说，将提供启动Dr. Dr.的技术培训和职业心态。 维尔（Velle）作为一项独立研究人员的职业生涯，研究计划的重点是纳格勒（Naegleria）的迁移。