Nucleation and Dissolution Mechanism Underlying ALS/FTLD-linked FUS Condensates

项目总结 融合于肉瘤(Fus)是一种rna结合蛋白，可以很容易地进行液-液相分离。 (LLP)在核内发挥其应有的功能。FUS突变和/或细胞应激导致 FU从细胞核到胞浆的错误定位和LLP的异常，导致有毒物质的形成 凝胶状或固体状的集合体比野生型(WT)FUS更具凝胶状或固体状，其冷凝物具有动态液体- 就像房产一样。突变的fus的有毒聚集体是年龄依赖性神经退行性疾病的标志。 如肌萎缩侧索硬化症(ALS)和额颞叶痴呆(FTLD) 由患者运动神经元的渐进性丧失和最终死亡引起。我建议确定机制 导致运动神经元死亡和疾病的细胞毒性FUS聚集体的形成 进步。具体地说，我建议利用单分子技术来研究成核和 FU聚集背后的溶解机制和驱动FU LLP的潜在相互作用 对与疾病相关的突变体感到不安。目标1将量化FUS在细胞中的寡聚状态 WT、胁迫和突变条件下的单分子下拉(SiMPull)分析。这一目标将考验 多个ALS/FTLD连锁的FUS突变多种形式的应激对FUS齐聚的影响 本地化依赖的方式。AIM 2将利用体外成核和溶出试验来比较 WT与突变的FUS低聚物的成核模式，并将确定FUS的作用机制 凝析油组件。此外，AIM 2将测试维护FUS凝析油所需的相互作用 用一组会干扰疏水性、静电性和核糖核酸的溶出剂进行溶解分析 相互作用，以研究各自相互作用的损失对FUS低聚物的影响。AIM 3将调查 FUS-RNA相互作用通过确定RNA序列、长度和结构在FUS成核中的作用 对于FUS凝析油的成核和维持是必要的，并将识别在 ALS/FTLD连锁突变体FUS冷凝物。这项建议得到了来自三个国家的支持 合作者(见支持函)，他们都是肌萎缩侧索硬化症、有限合伙人方面的专家，或者可以提供技术支持。这个 该奖项下计划的活动包括协作、专业发展机会、参与 参加科学会议，在实验室指导本科生，提高科学水平 沟通和批判性思维能力将使我成功地完成博士学位，并为我的 从事博士后工作，后来在学术界取得事业。这项提议将使我能够继续我的长期- 学期的职业目标，将利用单分子技术来更好地从机理上理解 ALS/FTLD连锁的FUS突变体可防止疾病进展。

PROJECT SUMMARY Fused in sarcoma (FUS) is an RNA binding protein which can readily undergo liquid-liquid phase separation (LLPS) to perform its proper functions in the nucleus. Mutations in FUS and/or cellular stress lead to mislocalization of FUS from the nucleus to the cytosol and aberrant LLPS, leading to the formation of toxic aggregates that are more gel-like or solid-like than wild type (WT) FUS whose condensates have dynamic liquid- like properties. Toxic aggregates of mutant FUS is a hallmark of age-dependent neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD) which are characterized by a progressive loss of motor neurons and eventual death in patients. I propose to identify mechanisms underlying the formation of cytotoxic FUS aggregates which contribute to the death of motor neurons and disease progression. Specifically, I propose to utilize single molecule techniques to investigate the nucleation and dissolution mechanisms underlying FUS aggregation and the underlying interactions driving FUS LLPS which become perturbed in disease-linked mutants. Aim 1 will quantify the oligomerization status of FUS in cells under WT, stressed and mutant conditions using single molecule pulldown (SiMPull) analysis. This aim will test the impact of multiple ALS/FTLD-linked FUS mutations multiple forms of stress on FUS oligomerization in a localization-dependent manner. Aim 2 will utilize in vitro nucleation and dissolution assays to compare the nucleation pattern of WT versus mutant FUS oligomers and will identify the mechanism of action underlying FUS condensate assembly. In addition, aim 2 will test the interactions necessary to maintain FUS condensates using dissolution assays with a panel of various dissolving agents that will perturb hydrophobic, electrostatic, and RNA interactions, to investigate the effects of loss of respective interactions on FUS oligomers. Aim 3 will investigate the role of FUS-RNA interactions in FUS nucleation by determining the RNA sequences, lengths, and structures necessary for nucleating and maintaining FUS condensates, and will identify interactions that are disrupted in ALS/FTLD-linked mutant FUS condensates. This proposal is strengthened by contributions from three collaborators (see support letters), all of whom are experts on ALS, LLPS, or can provide technical support. The activities planned under this award including collaborations, professional development opportunities, engaging and participating in scientific conferences, mentoring undergraduates in the lab, and improving scientific communication and critical thinking skills will allow me to successfully complete my PhD and prepare me for a postdoctoral position and later, to attain a career in academia. This proposal will allow me to pursue my long- term career goal and will utilize single molecule techniques to gain a better mechanistic understanding of ALS/FTLD-linked FUS mutants to prevent disease progression.