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”福氏耐格里阿米巴（Naegleria fowleri）引起的疾病死亡率高达97%。电流 治疗方法并不可靠，而且有可能产生严重的副作用，包括脑损伤。因为细胞增殖是 对于疾病进展至关重要，耐格里属的微管网络在进化上已经从 在人类中，靶向有丝分裂纺锤体是开发有效治疗方法的一种有前途的策略， 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， 耐格里属有丝分裂纺锤体，阻碍了合理治疗的进展。特别是，动态 在其他细胞中，微管周转对于组装双极纺锤体是至关重要的，但尚不清楚在何种程度上 耐格里属纺锤体依赖于微管动力学。一个主要的障碍是， 在其他物种中的动力学对耐格里属的分歧微管蛋白无效。此外，虽然微管 马达蛋白在不同物种的纺锤体组装中起着关键作用， 奈格里藻纺锤体内的微管马达是完全未知的。缺乏关于 微管周转和分子马达对耐格里藻纺锤体组织的作用机制 限制了确定抗菌剂开发的关键蛋白质和过程。 这项提案将通过检验微管动力学和细胞周期的假设来解决这一知识缺口。 分子马达都有助于耐格里属纺锤体组织。确定微管的作用 动力学，微管将被稳定与一类抑制剂，最近被证明可以阻止奈格莱里亚 细胞分裂，以及对纺锤体组织的影响将用超分辨率测量 显微镜比较未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。为了确定纺锤体中分子马达的功能， 在细胞分裂过程中上调将通过有丝分裂同步的耐格里属的RNA测序来鉴定 cultures.这些马达将被击倒，并通过显微镜对表型进行评分。拟建项目 将提供全面的培训，以准备申请人的职业生涯作为一个独立的调查员。与 来自赞助商的支持，谁是耐格里专家，和共同赞助商，谁有几十年的研究经验， 细胞分裂，申请人将学习：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细胞增殖的影响。 耐格里属纺锤体，并将确定纺锤体相关的分子马达在纺锤体组织中的作用， 为未来治疗耐格里属引起的毁灭性疾病提供了新的靶点。