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: 10657784
• 负责人: Katrina Velle
• 金额: $ 4.85万
• 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 2023-12-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10657784
• 关键词: Actins; Advisory Committees; Amoeba genus; Animals; Architecture; Automobile Driving; Behavior; Biochemistry; Biological; Biological Assay; Biological Models; Biology; Brain; Bulla; Cell physiology; Cells; Cellular biology; Cicatrix; Complement; Complex; Cytoskeleton; Defect; Dictyostelium discoideum; Disease; Drug Targeting; Eating; Electron Microscopy; Encephalitis; Ensure; Environment; Eukaryota; Foundations; Goals; 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; Postdoctoral Fellow; Property; Proteins; Pyrenes; Regulation; Research; Research Personnel; Signal Transduction; Speed; Surface; Techniques; Testing; Thinness; Total Internal Reflection Fluorescent; Training; Visualization; WASP protein; Wasps; Work; Yeasts; brain tissue; career; cell behavior; cell motility; cranium; defined contribution; experimental study; insight; knock-down; light microscopy; member; migration; nanoscale; pathogen; programs; recruit; skills; 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.

项目总结/摘要 虽然肌动蛋白在整个真核细胞中高度保守，但用于调节其组装和 拆卸在不同的门中是不同的。精确定时和定位的肌动蛋白组装几乎协调了每一个细胞 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实验中有了一些详细的定义， 细胞，这是未知的，如果不同的真核生物病原体使用相同的规则。这项建议会 解决的假设，有保守的原则，细胞迁移的调查耐格里， 在十亿年前从“酵母到人类”的谱系中分化出来。具体来说，这项工作将定义 细胞骨架对“食脑阿米巴”福氏耐格里阿米巴细胞爬行的贡献：一种病原体 这种病毒会爬进大脑，引起一种致命的脑膜炎， 治疗。Velle博士最初的博士后研究使用了常用的非致病性模型系统 Gruberi耐格里线虫强调了运动的普遍规则的潜力; gruberi在平坦的表面上爬行， 由称为Arp 2/3复合物的蛋白质组装的薄的、基于肌动蛋白的突起。这种基于肌动蛋白和Arp 2/3的 这一机制也是动物细胞如何在平面上爬行的。然而，在实验室条件之外，细胞 很少--如果有的话--能在平坦、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， 在复杂的、限制性的环境中爬行时的运动性，但这尚未在耐格里属中进行过测试。 因为N.福勒里爬行通过狭窄的通道，在头骨和大脑，尽管没有已知的 趋化性信号，解剖细胞迁移在限制性环境中是理解疾病必不可少的。 因此，目标1将在细胞行为水平上确定限制下细胞爬行的机制。 目标2将集中在细胞中的肌动蛋白网络;而突起驱动N。Gruberi平面迁移 看起来类似于动物细胞，使用铂离子电子显微镜定义肌动蛋白结构 （PREM）将揭示超微结构是否保守。这一目标还将提供关键培训， 博士Velle在光学显微镜下的背景。PREM的世界专家Svitkina博士将提供这一培训， 科学顾问委员会的成员目标3将使用生物化学-申请人没有的技术 之前的培训，以检查Arp 2/3复合物激活的上游机制。维尔医生招募了 博士布鲁斯古德，专家肌动蛋白生物化学家，为这项培训。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培训 和生物化学将确保申请人熟练掌握成功所需的技术。维尔博士还 我招募了肌动蛋白专家马特·韦尔奇博士，在肌动蛋白和变形虫方面有专长的梅格·泰特斯博士，以及吉姆· 莫里斯是N. fowleri女士向她的科学咨询委员会提供科学和职业指导。 总的来说，拟议的工作将提供技术培训，并推出博士所需的职业导师。 Velle的职业生涯是一名独立调查员，其研究项目主要集中在Naegleria的迁移。