In situ imaging of the aging-induced structural and stoichiometric degradation of the nuclear pore complex and nuclear periphery

老化引起的核孔复合体和核外围的结构和化学计量降解的原位成像

• 批准号: 10740706
• 负责人: Digvijay Singh
• 金额: $ 10.8万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10740706
• 关键词: Advisory Committees; Affect; Age; Aging; Architecture; Award; Bacteriophage T4; Biochemistry; Cell Aging; Cell physiology; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complement; Complex; Cryoelectron Microscopy; Cytoplasm; Deterioration; Development; Disease; Doctor of Philosophy; Electron Beam Tomography; Environment; Enzymes; Foundations; Frequencies; Functional disorder; Gene Expression Regulation; Goals; Health; Image; Imaging Techniques; Immobilization; In Situ; In Vitro; Investigation; Ions; Leadership; Link; Maps; Mentors; Mentorship; Modality; Modeling; Molecular; Morbidity - disease rate; Motor; Nerve Degeneration; Neurons; Nuclear; Nuclear Envelope; Nuclear Pore Complex; Nucleoplasm; Organism; Paper; Phase; Photobleaching; Play; Postdoctoral Fellow; Process; Protein Isoforms; Proteins; Publications; Reporting; Research; Resolution; Sampling; Science; Structure; Techniques; Therapeutic; Time; Training; Transcriptional Regulation; Work; Yeasts; aged; career; data integration; electron tomography; imaging modality; improved; in situ imaging; molecular assembly/self assembly; molecular imaging; molecular modeling; novel; nucleocytoplasmic transport; particle; programs; reconstitution; single molecule; skills; stoichiometry; structural biology; synthetic biology; trafficking; yeast genetics

PROJECT SUMMARY Ultrastructural molecular/sub-molecular level aging-induced degradation in cellular assemblies makes them dys- functional leading to cellular dysfunction that causes and exacerbates the aging-related problems. However, no investigation has been carried out to explore such degradations due to the lack of imaging modalities that are sen- sitive enough to capture the sub-molecular level features inside the cells in situ. One such assembly is the Nuclear Pore complex which creates a conduit in the nuclear membrane essential for cytoplasm-nucleoplasm trafficking, chromatin silencing, transcriptional control, and other vital cellular processes. NPC is a highly dynamic assembly formed of extremely long-lived aging-affected proteins, whose composition, structure, and function deteriorate with aging. This deterioration then negatively impacts their critical cellular functions, causing a decay in the health and function of the cells, which is especially relevant for non-dividing, non-rejuvenating, and aging-prone cells like neurons, thereby contributing to neurodegeneration. Cryo-focused ion beam milling and electron tomography (cryo-FIB-ET) imaging is a novel and emerging imaging modality that allows us to peek into the cells at the sub-molecular level, enabling in situ structural biology. Complementing cryo-FIB-ET imaging, single-molecule imaging can be used to explore the identification of the building blocks of massive cellular assemblies and also the molecular mechanism of its assembly. Using cryo-FIB-ET, Dr. Singh recently identified the in situ architecture of the NPC (Cell (2022)). He has also contributed to the development and application of cryo-FIB-ET imaging with contributions to 4 publications in his postdoc. During Ph.D., Dr. Singh trained in single-molecule imaging to study the molecular mechanism of CRISPR enzymes and T4-bacteriophage motor with 11 publications, including 6 as first/co-first. By leveraging his dual expertise in cryo-FIB-ET and single-molecule imaging and a broad collaboration effort within UCSD, around UCSD and with his collaborator and co-mentor at Rockfeller, Dr. Singh will determine: • The structures and isoforms of aged NPCs to map its aging-accumulated degradations (K99 phase). • Aging-induced changes in composition of NPC and its building blocks (K99 to R00 transition). • The assembly process of the NPC to understand how aging affects it (R00 phase). Dr. Singh will receive the needed training under the mentorship of Drs. Elizabeth Villa and Michael Rout. These mentors have demonstrated excellence in research leadership and have trained multiple trainees who have transitioned to independent fulfilling careers in sciences. In addition, an excellent team of collaborators and advisory committee (Drs. Andrej Sali, Nan Hao, Elizabeth Villa, Michael Rout) has been assembled to assist Dr. Singh’s research and provide additional training and career support through the K99-R00 phase. The R00 Award phase will set the foundation for Dr. Singh’s long-term career goals of establishing a rigorous research program that seeks to merge two powerful imaging modalities to understand the cellular level changes caused by aging at the molecular level.

项目总结 超微结构分子/亚分子水平的老化诱导的细胞组件的降解使其动态变化。 功能性导致细胞功能障碍，从而导致并加剧与衰老相关的问题。然而，没有 已经开展了调查以探索这种退化，这是由于缺乏能够检测到的成像模式 阳性程度足以在细胞内原位捕捉到亚分子水平的特征。其中一个这样的大会就是核 在核膜上形成对细胞质-核质运输至关重要的管道的孔道复合体， 染色质沉默、转录控制和其他重要的细胞过程。NPC是一个高度动态的程序集 由寿命极长的受衰老影响的蛋白质组成，其组成、结构和功能随着 衰老。这种恶化会对他们重要的细胞功能产生负面影响，导致健康状况恶化。 以及细胞的功能，这与不分裂、不返老还童和易衰老的细胞特别相关 像神经元一样，从而导致神经退化。低温聚焦离子束球磨和电子断层扫描 (Cryo-FIB-ET)成像是一种新颖的新兴成像方式，它使我们能够窥探细胞 亚分子水平，使原位结构生物学成为可能。补充冷冻-FIB-ET成像，单分子 成像可以用来探索大量细胞组装的构建块的识别，还 其组装的分子机制。使用冷冻-FIB-ET，辛格博士最近发现了原位 全国人大的架构(单元(2022年))。他还为开发和应用 冷冻-FIB-ET成像，在他的博士后发表了4篇论文。在博士期间，辛格博士接受了 单分子成像技术研究CRISPR酶和T4-噬菌体马达的分子机制 11份出版物，其中6份为第一/联合第一。通过利用他在冷冻-FIB-ET和单分子方面的双重专长 在UCSD内部、围绕UCSD以及与他的合作者和共同导师进行广泛的合作 罗克菲勒，辛格博士将确定： ·老化的NPC的结构和异构体，以绘制其老化-累积降解(K99相)。 ·衰老引起鼻咽癌及其构成成分的变化(从K99到R00的转变)。 ·NPC的组装过程，以了解老化对其的影响(R00阶段)。 辛格博士将在伊丽莎白·维拉博士和迈克尔·鲁特博士的指导下接受必要的培训。 这些导师在研究领导方面表现出卓越的表现，并培训了多名学员，这些学员 在科学方面过渡到独立的、有成就感的职业。此外，一个优秀的合作者团队和 咨询委员会(安德烈·萨利博士、南浩博士、伊丽莎白·维拉博士、迈克尔·鲁特博士)已经成立，以协助Dr。 辛格的研究，并通过K99-R00阶段提供额外的培训和职业支持。R00奖 该阶段将为辛格博士的长期职业目标奠定基础，即建立一个严格的研究计划 寻求将两种强大的成像模式结合起来，以了解由衰老引起的细胞水平变化 分子水平。