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

项目摘要 超微结构分子/亚分子水平的老化诱导的细胞组装体的降解，使他们dys- 导致细胞功能障碍，引起并加剧衰老相关问题。但没有 已经进行了调查，以探索由于缺乏感测到的成像模式而导致的这种退化， 足够灵敏以原位捕获细胞内的亚分子水平特征。其中一个组件是核 在核膜中形成细胞质-核质运输所必需的通道的孔复合体， 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而退化。 衰老这种恶化然后负面影响他们的关键细胞功能，导致健康衰退 和细胞的功能，这与非分裂、非再生和易老化的细胞特别相关 就像神经元一样，从而导致神经退化。低温聚焦离子束铣削和电子层析成像 （cryo-FIB-ET）成像是一种新的新兴成像方式，使我们能够窥视到细胞在 亚分子水平，实现原位结构生物学。补充冷冻FIB-ET成像，单分子 成像可用于探索大规模细胞组装的构建块的识别， 其组装的分子机制。辛格博士最近利用冷冻FIB-ET技术， NPC的架构（Cell（2022））。他还为开发和应用 cryo-FIB-ET成像，在博士后期间发表了4篇论文。在读博士期间，辛格博士曾在 单分子成像研究CRISPR酶和T4噬菌体马达的分子机制， 11篇出版物，包括6篇首次/共同首次出版物。通过利用他在冷冻FIB-ET和单分子 成像和广泛的合作努力，在UCSD内，围绕UCSD，并与他的合作者和共同导师在 洛克菲勒，辛格博士将决定： ·老化NPC的结构和亚型，以绘制其老化累积降解（K99阶段）。 ·衰老诱导的NPC组成及其结构单元的变化（K99至R 00转变）。 ·NPC的组装过程，以了解老化对其的影响（R 00阶段）。 博士辛格将在伊丽莎白·维拉和迈克尔·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色，并培训了多名学员， 过渡到独立的科学成就事业。此外，一个优秀的合作者团队和 咨询委员会（Andrej Sali博士，Nan Hao博士，Elizabeth Villa博士，Michael Rout博士）已成立，以协助Dr. 辛格的研究，并通过K99-R 00阶段提供额外的培训和职业支持。R 00奖 这一阶段将为辛格博士建立一个严格的研究计划的长期职业目标奠定基础 该研究试图将两种强大的成像方式结合起来，以了解衰老引起的细胞水平变化， 分子水平。

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.