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

项目摘要 肉瘤融合蛋白（FUS）是一种RNA结合蛋白，可容易地进行液-液相分离 （LLPS）在细胞核中发挥其应有的功能。FUS突变和/或细胞应激导致 FUS从细胞核到胞质溶胶的错误定位和异常LLPS，导致形成毒性 比野生型（WT）FUS更凝胶样或固体样的聚集体，其冷凝物具有动态液体- 比如财产突变FUS的毒性聚集是年龄依赖性神经退行性疾病的标志 例如肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTLD）， 运动神经元的逐渐丧失最终导致患者死亡。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 进展具体来说，我建议利用单分子技术来研究成核和 FUS聚集的溶解机制和驱动FUS LLPS的潜在相互作用， 在疾病相关的突变体中变得不安。目的1将量化在细胞内FUS的寡聚化状态， 使用单分子下拉（SiMPull）分析的WT、应激和突变体条件。这一目标将考验 多种ALS/FTLD-连锁FUS突变的影响多种形式的应激对FUS寡聚化的影响 定位依赖的方式。目的2将利用体外成核和溶出试验来比较 WT与突变FUS寡聚体的成核模式，并将确定FUS的作用机制 冷凝水组件。此外，aim 2将测试维持FUS冷凝物所需的相互作用， 使用一组各种溶解剂进行溶解测定，这些溶解剂将干扰疏水性、静电性和RNA 相互作用，以研究丧失各自的相互作用对FUS寡聚体的影响。目标3将进行调查 通过确定RNA序列、长度和结构，FUS-RNA相互作用在FUS成核中的作用 成核和维持FUS冷凝物所必需的，并将确定在FUS冷凝物中被破坏的相互作用。 ALS/FTLD连锁突变FUS缩合物。这一建议得到了三个国家的支持。 合作者（见支持信），他们都是ALS，LLPS的专家，或者可以提供技术支持。的 该奖项下计划的活动包括合作，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我的博士学位，并为我准备一个 博士后的位置，后来，在学术界获得职业生涯。这个提议将使我能够继续我长期的- 长期的职业目标，并将利用单分子技术，以获得更好的机械理解， ALS/FTLD连锁的FUS突变体，以防止疾病进展。

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.