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：
10668388
负责人：
Nathalie Ashley Djaja
金额：
$ 4.77万
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 2024-11-15
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10668388
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 Genetic Transcription Goals Hydrophobicity In Vitro Lead Length Letters Link Liquid substance Maintenance Measures Mentors Messenger RNA Molecular Motor Neurons Mutate Mutation Neurodegenerative Disorders Nuclear Nuclear RNA Pancreatic ribonuclease Pathologic Patients Pattern Phase Physical condensation Play Positioning Attribute Postdoctoral Fellow Process Property Proteins RNA RNA Binding RNA Sequences RNA Splicing RNA metabolism RNA-Binding Proteins Reagent Reporting Research Role Series Sodium Chloride Solid Stress Structure Techniques Testing Therapeutic Translations Viscosity age related biological adaptation to stress career cellular imaging cytotoxic experimental study frontotemporal lobar dementia amyotrophic lateral sclerosis fused in sarcoma improved in vivo insight liquid dynamics mRNA Export monomer motor disorder 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.

项目摘要在肉瘤中融合（FUS）是一种RNA结合蛋白，可以容易地进行液态液相分离（LLP）在细胞核中执行其适当的功能。 FUS和/或细胞应激中的突变导致从细胞核到细胞质和异常LLP的FUS错误定位，导致有毒的形成比野生型（wt）的凝胶状的聚集体，其冷凝水具有动态液体 - 喜欢属性。突变FUS的有毒骨料是年龄依赖性神经退行性疾病的标志例如肌萎缩性侧索硬化症（ALS）和额颞叶痴呆症（FTLD）的特征通过运动神经元的进行性丧失和患者最终死亡。我建议确定机制基本的细胞毒性FUS聚集体的形成，有助于运动神经元和疾病的死亡进展。具体而言，我建议利用单分子技术研究成核和溶解机制的基础机器人的固定机制和驱动FUS LLP的潜在相互作用，这些相互作用在疾病连接的突变体中受到干扰。 AIM 1将量化细胞中FUS的寡聚状态 WT，使用单分子下拉（SIMPULL）分析，应力和突变条件。这个目标将测试多种ALS/FTLD连接的FUS突变的影响多种形式的应力对A中的FUS寡聚化的影响定位依赖性方式。 AIM 2将利用体外成核和溶解测定来比较 WT与突变型FUS低聚物的成核模式，将确定FUS基础作用机理冷凝水组件。此外，AIM 2将测试使用使用FUS冷凝物所需的相互作用用各种溶解剂的面板溶解测定，这些溶解剂会扰动疏水，静电和RNA 相互作用，以研究各自相互作用对FUS低聚物的影响。 AIM 3将调查通过确定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护FUS冷凝物所必需的，并将确定被破坏的相互作用 ALS/FTLD连接的突变型FUS冷凝物。三个提议得到了加强合作者（请参阅支持信），他们都是ALS，LLP的专家，或者可以提供技术支持。这根据该奖项计划的活动，包括合作，专业发展机会，参与并参加科学会议，指导实验室的本科生并改善科学沟通和批判性思维能力将使我能够成功完成博士学位，并为我做好准备博士后职位，后来是在学术界的职业。该建议将使我能够追求我的长期任期职业目标，并将利用单分子技术来获得更好的机械理解 ALS/FTLD连接的FUS突变体可预防疾病进展。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Nucleation and dissolution mechanism underlying amyotrophic lateral sclerosis/frontotemporal lobar dementia-linked fused in sarcoma condensates.

DOI：
10.1016/j.isci.2023.106537
发表时间：
2023-04-21
期刊：
ISCIENCE
影响因子：
5.8
作者：
Djaja, Nathalie A.;Chang, Matthew T.;Morris, Freya R.;Morris, Vivian M.;Ganser, Laura R.;Myong, Sua
通讯作者：
Myong, Sua

Protocol for single-molecule pull-down of fluorescently tagged oligomers from cell lysates.