Defining mechanisms of mitotic spindle organization in Naegleria

耐格里虫有丝分裂纺锤体组织的定义机制

• 批准号: 10537005
• 负责人: Andrew Steven Kennard
• 金额: $ 6.72万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10537005
• 关键词: Address; Affect; Amoeba genus; Animals; Architecture; Automobile Driving; Biochemical; Biological; Biology; Brain; Brain Injuries; Case Fatality Rates; Category B pathogen; Cell Proliferation; Cell division; Cells; Cellular Structures; Cellular biology; Chromosomes; Data; Development; Disease; Disease Progression; Drug Targeting; Eating; Electron Microscopy; Ensure; Fatality rate; Future; Gene Expression; Gene Proteins; Genes; Goals; Heat-Shock Response; High-Throughput Nucleotide Sequencing; Human; Image; Infection; Knowledge; Lead; Learning; Length; Measurement; Measures; Mentors; Methods; Microscopy; Microtubule Stabilization; Microtubule stabilizing agent; Microtubules; Mitosis; Mitotic; Mitotic spindle; Molecular; Molecular Motors; Morphology; Motor; Naegleria; Naegleria fowleri; National Institute of Allergy and Infectious Disease; Organism; Pharmaceutical Preparations; Phenotype; Play; Population; Process; Proteins; Protocols documentation; Quantitative Microscopy; RNA Splicing; Research Personnel; Resolution; Risk; Role; Running; Sequence Analysis; Source; Structure; Techniques; Testing; Therapeutic; Training; Tubulin; antimicrobial; base; career; career development; effective therapy; experience; inhibitor; knock-down; side effect; skill acquisition; success; temporal measurement; therapeutic development; therapeutically effective; therapy development; transcriptome sequencing; transcriptomics

Project Summary The “brain-eating amoeba” Naegleria fowleri causes a disease with a 97% fatality rate. Current treatments are not reliable and risk significant side effects, including brain damage. Because cell proliferation is essential for disease progression, and the microtubule network in Naegleria has evolutionarily diverged from that in humans, targeting the mitotic spindle is a promising strategy to develop effective therapeutics with limited side effects. However, we lack key information about the basic cell biological mechanisms that organize the Naegleria mitotic spindle, hampering progress towards rational therapies. In particular, dynamic microtubule turnover is critical in other cells for assembling a bipolar spindle, but it is not known to what extent the Naegleria spindle relies on microtubule dynamics. A major obstacle is that inhibitors that block microtubule dynamics in other species are ineffective against Naegleria’s divergent tubulins. Further, while microtubule motor proteins play key roles in assembling spindles from diverse species, the function or even the identity of microtubule motors within the Naegleria spindle is completely unknown. Lack of knowledge about the mechanistic contributions of microtubule turnover and molecular motors to Naegleria spindle organization constrains identifying key proteins and processes to target for antimicrobial development. This proposal will address this knowledge gap by testing the hypothesis that microtubule dynamics and molecular motors both contribute to Naegleria spindle organization. To identify the role of microtubule dynamics, microtubules will be stabilized with a class of inhibitors that was recently shown to block Naegleria cell division, and the effect on the organization of the spindle will be measured with super-resolution microscopy. Comparing untreated and drug-treated spindles at different mitotic stages will reveal which stages require microtubule turnover. To determine the function of molecular motors in the spindle, motor genes upregulated during cell division will be identified with RNA sequencing of mitotically synchronized Naegleria cultures. These motors will be knocked down, and phenotypes scored by microscopy. The proposed project will provide comprehensive training to prepare the applicant for a career as an independent investigator. With support from a sponsor, who is a Naegleria expert, and a co-sponsor, who has decades of experience studying cell division, the applicant will learn: new experimental techniques, such as super-resolution microscopy and high-throughput sequencing; new conceptual approaches, including the biology of microtubules and mitosis; and career development skills. This proposal will determine the impact of microtubule stabilization on the Naegleria spindle and will identify the role of spindle associated molecular motors in spindle organization, providing new targets for future treatments for the devastating disease caused by Naegleria.

项目摘要 食脑阿米巴引起的福氏奈格勒杆菌致死率高达97%。当前 治疗方法不可靠，有可能产生严重的副作用，包括脑损伤。因为细胞增殖是 对于疾病的发展是必不可少的，而奈格勒属的微管网络在进化上已经偏离了 在人类中，以有丝分裂纺锤体为靶点是开发有效疗法的一种很有前途的策略 副作用有限。然而，我们缺乏关于组织的基本细胞生物学机制的关键信息 Naegleria有丝分裂纺锤体，阻碍了理性治疗的进展。尤其是动态的 微管的更新在其他细胞中对于组装双极纺锤体是至关重要的，但具体到什么程度尚不清楚。 Naegleria纺锤体依赖于微管动力学。一个主要的障碍是阻断微管的抑制剂 其他物种的动态对Naegleria的发散微管蛋白无效。此外，虽然微管 马达蛋白在组装不同物种的纺锤体、功能甚至识别纺锤体方面起着关键作用。 Naegleria纺锤体内的微管马达是完全未知的。缺乏关于这方面的知识 微管周转和分子马达对Naegleria纺锤体组织的机械贡献 限制识别关键蛋白质和过程，使其成为抗菌药物开发的目标。 这一建议将通过检验微管动力学和微管动力学的假设来解决这一知识差距 分子马达对Naegleria纺锤体的组织都有贡献。确定微管的作用 动力学，微管将被一类最近被证明能阻断Naegleria的抑制剂稳定下来 细胞分裂，对纺锤体组织的影响将用超分辨率测量 显微镜。比较未经处理和药物处理的纺锤体在不同的有丝分裂阶段将揭示哪些阶段 需要微管周转。为了确定分子马达在纺锤体中的功能，马达基因 在细胞分裂过程中上调的基因将通过有丝分裂同步的Naegleria的RNA测序来鉴定 文化。这些马达将被拆卸，并通过显微镜对表型进行评分。拟议中的项目 将提供全面的培训，为申请人成为一名独立调查员做好准备。使用 来自赞助商的支持，他是Naegleria专家，也是联合赞助商，他有几十年的研究经验 细胞分裂，申请者将学习：新的实验技术，如超分辨率显微镜和 高通量测序；新的概念方法，包括微管和有丝分裂的生物学； 和职业发展技能。这项建议将确定微管稳定对血管内皮细胞 Naegleria纺锤体，并将确定纺锤体相关分子马达在纺锤体组织中的作用， 为未来治疗由Naegleria引起的毁灭性疾病提供新的目标。