Defining actin-based mechanisms driving basic cell functions and pathogenic behaviors in Naegleria

定义基于肌动蛋白的机制，驱动耐格里虫的基本细胞功能和致病行为

• 批准号: 10213600
• 负责人: Katrina Velle
• 金额: $ 7.04万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-25 至 2023-08-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10213600
• 关键词: Actins; Actomyosin; Address; Amoeba genus; Animals; Automobile Driving; Behavior; Biology; Brain; Caliber; Category B pathogen; Cell Survival; Cell division; Cell membrane; Cell physiology; Cells; Cellular biology; Complement; Complex; Core Facility; Cytokinesis; Cytometry; Cytoskeleton; Data; Defect; Disease; Drug Targeting; Eating; Encephalitis; Ensure; Environment; Equilibrium; Eukaryota; F-Actin; Fatality rate; Flow Cytometry; Foundations; Future; Gene Expression Profiling; Gene Targeting; Goals; Health; Human; Image; Impairment; Individual; Infection; Measures; Mentorship; Microscopy; Microtubules; Mission; Mitotic; Molecular; Myosin ATPase; Myosin Type II; Naegleria; Naegleria fowleri; National Institute of Allergy and Infectious Disease; Organelles; Organism; Osmolar Concentration; Osmotic Pressure; Pathogenesis; Pathogenicity; Phenotype; Play; Population; Postdoctoral Fellow; Process; Protein Family; Proteins; Pump; Research; Research Personnel; Research Training; Resolution; Resource Development; Resources; Role; Signal Transduction; Solid; Testing; Training; Transcript; Translations; United States National Institutes of Health; Universities; Vacuole; Virulence; Water; Work; base; career; career development; cell motility; cellular imaging; chemokine; driving force; migration; single cell analysis; small molecule inhibitor; success; therapeutic development

Project Summary The “brain-eating amoeba” Naegleria fowleri is an NIAID Priority Category B Pathogen that carries a 95% fatality rate, yet the mechanisms underlying its basic biology and pathogenic behaviors remain largely unstudied. Because understanding the cell biology of Naegleria is critical to the development of therapeutics, my long-term goal is to define the cellular and molecular basis of Naegleria pathogenesis. Unlike human cells, from which Naegleria diverged 1-2 billion years ago, these amoebae do not possess cytoplasmic microtubules. This suggests that the actin cytoskeleton, assembled by the Arp2/3 complex and formin family proteins, is the primary driving force for many cellular processes essential to cell survival and pathogenesis. Therefore, my overall objective in this application is to determine how actin cytoskeletal rearrangements promote cell motility, which is important for establishing infection, contractile vacuole pumping, which is required for surviving osmotic pressure, and cell division, which is critical for robust colonization in the brain. To achieve this objective, I propose the following specific aims: (Aim 1) determine the Arp2/3 complex activators driving cell motility, (Aim 2) define the formin-based mechanisms governing contractile vacuole dynamics, and (Aim 3) identify the actin nucleators and molecular mechanisms responsible for cytokinesis. I will address these aims using orthogonal cellular perturbations (small molecule inhibitors to impair the Arp2/3 complex and formin family proteins, as well as individual gene targeting using morpholinos) and environmental perturbations by changing external osmotic pressure. I will measure phenotypes of single cells in detail with microscopy, and complement this by collecting quantitative data on cell populations with flow cytometry and gene expression assays. Because the cell biology underlying pathogenesis and basic functions in Naegleria is severely understudied, defining how actin orchestrates motility, division, and contractile vacuole dynamics is critical for uncovering drug targets to treat these deadly infections. Completing this project will not only advance the mission of the NIH, but it will also prepare me for a career as an independent investigator at a research-intensive university studying the cell biology of Naegleria. The career development resources available to postdocs and the excellent core facility trainings at UMass create an ideal institutional environment for my training. Further, the mentorship of Dr. Fritz- Laylin, an expert on Naegleria biology, and Dr. Wadsworth, who specializes in cell division, will ensure the success of this project as well as my transition to a career as an independent researcher.

项目摘要 “食脑阿米巴”福氏奈格勒杆菌是一种NIAID优先B类病原体，其致死率为95% 然而，其基本生物学和致病行为背后的机制仍未得到很大程度的研究。 因为了解Naegleria的细胞生物学对治疗学的发展至关重要，我的长期 目的是明确Naegleria致病的细胞和分子基础。与人类细胞不同，人类细胞 奈格勒虫在10-20亿年前分化，这些阿米巴虫不具有细胞质微管。这 提示由Arp2/3复合体和Forin家族蛋白组装的肌动蛋白细胞骨架是主要的 许多细胞过程的驱动力，对细胞生存和致病至关重要。因此，我的总体 本应用的目的是确定肌动蛋白细胞骨架重排如何促进细胞运动，这是 对于建立感染很重要，收缩空泡泵送是渗透生存所必需的 压力和细胞分裂，这对大脑中强大的定植至关重要。为达致这个目标，我 提出以下具体目标：(目标1)确定驱动细胞运动的Arp2/3复合激活剂；(目标2) 定义基于福尔马林的控制收缩空泡动力学的机制，并(目标3)确定肌动蛋白 负责胞质分裂的核因子和分子机制。我将使用正交表解决这些目标 细胞扰动(也包括损害Arp2/3复合体和Forin家族蛋白的小分子抑制剂 作为使用吗啡的单个基因靶向)和通过改变外部渗透来进行环境扰动 压力。我将用显微镜详细测量单个细胞的表型，并通过收集 用流式细胞仪和基因表达分析对细胞群体进行定量数据。因为细胞生物学 Naegleria的潜在发病机制和基本功能研究严重不足，定义了肌动蛋白是如何 协调运动、分裂和收缩空泡动力学对发现治疗药物靶点至关重要 这些致命的感染。完成这一项目不仅将推进NIH的使命，而且还将 为我在一所研究密集的大学从事细胞研究的独立调查员的职业生涯做好准备 Naegleria的生物学。为博士后提供的职业发展资源和优秀的核心设施 在马萨诸塞州大学的培训为我的培训创造了理想的制度环境。此外，弗里茨博士的指导- Naegleria生物学专家Laylin和专门研究细胞分裂的Wadsworth博士将确保 这个项目的成功以及我向独立研究人员职业生涯的过渡。