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.

项目摘要 超微结构分子/亚分子水平衰老诱导的细胞组件降解使它们dys- 功能导致细胞功能障碍引起并加剧与衰老有关的问题。但是，不 由于缺乏参数的成像方式，已经进行了调查以探索这种降解 粘贴足以捕获细胞内部的亚分子水平特征。一个这样的大会是核 孔复合物在核膜中产生导管，这对于细胞质核核菌群的必不可少， 染色质沉默，转录控制和其他重要的细胞过程。 NPC是一个高度动态的组装 由非常长的衰老蛋白形成，其组成，结构和功能确定 老化。然后，该定义会对其关键细胞功能产生负面影响，从而导致健康衰减 细胞的功能，这与非分裂，非恢复和易于衰老的细胞特别相关 像神经元一样，从而导致神经变性。以低温为重点的离子束铣削和电子断层扫描 （Cryo-Fib-et）成像是一种新颖而新兴的成像方式，使我们能够窥视细胞 亚分​​子水平，实现原位结构生物学。补充冷冻纤维-ET成像，单分子 成像可用于探索大规模蜂窝组件的构件的识别以及 其组装的分子机制。 Singh博士使用Cryo-Fib-Et，最近确定了原位 NPC的体系结构（单元格（2022））。他还为开发和应用做出了贡献 Cryo-Fib-Et成像为他的博士后中的4个出版物做出了贡献。在博士学位期间，辛格博士接受了培训 单分子成像研究CRISPR酶的分子机制和带有T4-细菌的电动机的分子机制 11个出版物，包括第一个/联合首先。通过利用其双重专家在Cryo-Fib-Et和单分子中 UCSD，UCSD围绕UCSD以及他的合作者兼联合主管的成像和广泛的合作工作 摇滚乐手，辛格博士将确定： •老化NPC的结构和同工型绘制其老化含量的降解（K99相）。 •衰老引起的NPC组成及其构件的变化（K99至R00过渡）。 •NPC的组装过程，以了解衰老的影响（R00阶段）。 辛格博士将在Drs的心态下接受所需的培训。伊丽莎白·维拉（Elizabeth Villa）和迈克尔·鲁特（Michael Rout）。 这些导师在研究领导方面表现出卓越的表现，并培训了多位学员 过渡到科学中独立实现的职业。此外，一支优秀的合作者团队和 咨询委员会（Andrej Sali博士，Nan Hao，Elizabeth Villa，Michael Rout）已被聚集以协助博士。 辛格（Singh）的研究，并通过K99-R00阶段提供了其他培训和职业支持。 R00奖 阶段将为辛格博士建立严格的研究计划的长期职业目标奠定基础 试图合并两种强大的成像方式，以了解衰老引起的细胞水平变化 分子水平。