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

ALS/FTLD 连接的 FUS 缩合物的成核和溶解机制

基本信息

批准号：
10474309
负责人：
Nathalie Ashley Djaja
金额：
$ 4.68万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10474309
关键词：
Academia Amyotrophic Lateral Sclerosis Automobile Driving Award Biochemical Biological Assay Buffers Cell Nucleus Cells Cellular Stress Cessation of life Collaborations Communication Complex Critical Thinking Cytosol Dependence Development Disease Disease Progression Doctor of Philosophy Electrostatics Exhibits Frontotemporal Dementia Gel Goals Hydrophobicity In Vitro Lead Length Letters Link Liquid substance Maintenance Measures Mentors Messenger RNA Molecular Motor Neuron Disease Motor Neurons Mutate Mutation Neurodegenerative Disorders Nuclear Nuclear RNA Pancreatic ribonuclease Pathologic Patients Pattern Phase Play Positioning Attribute Process Property Proteins RNA RNA Binding RNA Sequences RNA metabolism RNA-Binding Proteins Reagent Reporting Research Role Series Sodium Chloride Solid Stress Structure Techniques Testing Therapeutic Viscosity age related base career cellular imaging cytotoxic experimental study frontotemporal lobar dementia-amyotrophic lateral sclerosis fused in sarcoma improved in vivo insight mutant prevent single molecule skills symposium undergraduate student

项目摘要

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.

项目概要肉瘤融合蛋白 (FUS) 是一种 RNA 结合蛋白，很容易进行液-液相分离（LLPS）在细胞核中发挥其适当的功能。 FUS 突变和/或细胞应激导致 FUS 从细胞核错误定位到细胞质和异常的 LLPS，导致有毒物质的形成比野生型 (WT) FUS 更具凝胶状或固体状的聚集体，其冷凝物具有动态液体- 喜欢属性。突变 FUS 的有毒聚集体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）患者运动神经元逐渐丧失并最终死亡。我建议确定机制细胞毒性 FUS 聚集体形成的基础，导致运动神经元死亡和疾病进展。具体来说，我建议利用单分子技术来研究成核和 FUS 聚集的溶解机制以及驱动 FUS LLPS 的潜在相互作用在与疾病相关的突变体中变得不安。目标 1 将量化以下细胞中 FUS 的寡聚状态使用单分子下拉 (SiMPull) 分析的 WT、应激和突变条件。这个目标将考验多种 ALS/FTLD 相关 FUS 突变的影响多种形式的应激对 FUS 寡聚化的影响依赖于本地化的方式。目标 2 将利用体外成核和溶解测定来比较 WT 与突变型 FUS 寡聚物的成核模式，并将确定 FUS 的作用机制凝结水组装。此外，目标 2 将测试维持 FUS 冷凝物所需的相互作用，使用使用一组会干扰疏水性、静电和 RNA 的各种溶解剂进行溶解测定相互作用，以研究 FUS 寡聚物各自相互作用丧失的影响。目标 3 将进行调查通过确定 RNA 序列、长度和结构，了解 FUS-RNA 相互作用在 FUS 成核中的作用是成核和维持 FUS 凝聚物所必需的，并且将识别在 ALS/FTLD 连接的突变体 FUS 凝聚物。三个人的贡献加强了该提案合作者（请参阅支持信函），他们都是 ALS、LLPS 方面的专家，或者可以提供技术支持。这该奖项计划的活动包括合作、专业发展机会、参与参加科学会议，指导实验室的本科生，提高科学水平沟通和批判性思维能力将使我能够成功完成我的博士学位，并为我的未来做好准备博士后职位，后来在学术界取得职业生涯。这个建议将使我能够追求我的长期目标术语职业目标，并将利用单分子技术来更好地理解 ALS/FTLD 连接的 FUS 突变体可预防疾病进展。