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.

项目概要/摘要 尽管肌动蛋白在真核生物中高度保守，但用于调节其组装和组装的机制 不同门的拆卸各不相同。精确定时和放置的肌动蛋白组装协调几乎所有细胞 过程，包括细胞迁移。虽然肌动蛋白驱动的细胞迁移已经在动物中得到了一些详细的定义 细胞中，尚不清楚不同的真核病原体是否使用相同的规则进行操作。该提案将 通过研究 Naegleria 来解决细胞迁移存在保守原理的假设， 十亿多年前就与“酵母到人类”的谱系分歧了。具体来说，这项工作将定义 细胞骨架对“食脑变形虫”福氏耐格里阿米巴细胞爬行的贡献：一种病原体 它爬入大脑并在大脑内引起一种致命的脑膜炎，目前还没有可靠的治疗方法 治疗。 Velle博士最初的博士后研究使用常用的非致病性模型系统 格鲁氏耐格里虫强调了通用运动规则的潜力； N. gruberi 在平坦的表面上爬行，使用 薄的、基于肌动蛋白的突起由称为 Arp2/3 复合体的蛋白质组装而成。这种肌动蛋白和 Arp2/3 为基础 机制也是动物细胞在平坦表面上爬行的方式。然而，在实验室条件之外，细胞 很少（如果有的话）在平坦、均匀的表面上爬行。众所周知，动物细胞会切换到不同的模式 在复杂、限制性的环境中爬行时具有运动能力，但这尚未在耐格里变形虫中进行过测试。 因为福氏猪笼草爬行穿过颅骨中的狭窄通道并进入大脑，尽管目前尚不清楚 趋化信号，剖析限制性环境中的细胞迁移对于了解疾病至关重要。 因此，目标1将在细胞行为水平上确定细胞在限制下爬行的机制。 目标 2 将重点关注细胞中的肌动蛋白网络；而突起则驱动格鲁伯里猪笼草在平坦表面上迁移 看起来与动物细胞相似，使用铂金复制品电子显微镜定义了肌动蛋白结构 (PREM) 将揭示超微结构是否保守。这一目标还将提供关键培训来补充 Velle 博士在光学显微镜方面的背景。世界 PREM 专家 Svitkina 博士将提供此培训 科学顾问委员会成员。目标 3 将使用生物化学——申请人没有这项技术 之前的培训——检查 Arp2/3 复合物激活的上游机制。 Velle博士已招募 肌动蛋白生物化学专家 Bruce Goode 博士参加了本次培训。因为细胞领域的领先实验室 迁移经常使用显微镜和体外肌动蛋白生物化学，建议在 PREM 中进行培训 生物化学将确保申请人熟练掌握成功所需的技术。 Velle 博士还 招募了肌动蛋白专家 Matt Welch 博士、肌动蛋白和变形虫专业知识的 Meg Titus 博士以及 Jim 博士 莫里斯是福氏猪笼草专家，她向她的科学顾问委员会提供科学和职业指导。 总的来说，拟议的工作将提供启动博士所需的技术培训和职业指导。 Velle 的职业生涯是作为一名独立调查员，其研究项目重点关注耐格里变形虫的迁徙。