The dynamic architecture of living cells: Uncovering intra-organelle events at nanoscale levels

项目总结 细胞功能是通过高度动态的细胞器的复杂调节和协调来维持的，这 它们本身包含多个组件和蛋白质组件，这些组件和蛋白质组件不断地一起发挥作用，促进 细胞动态平衡。然而，到目前为止，细胞生物学领域主要集中在细胞器动力学上 整个细胞器水平，或细胞器间膜接触部位的细胞器之间的串扰。在……里面 相比之下，发生在细胞器内细胞器内水平的分子事件，如在细胞器内 线粒体基质，以及这些事件的时间、分布和调节 未被探索，主要是因为之前缺乏高速超分辨率显微镜来探测这些 纳米级的问题。重要的是，填补这些缺失的片段将是细胞领域的关键 生物学通过实时动态绘制活细胞的细胞结构图，并推动我们的 在细胞器内水平上的细胞内平衡的知识。线粒体是高度关键的细胞器 调节细胞功能，并包含它们自己的mtDNA，该DNA在 线粒体基质，以及有助于促进有效折叠的线粒体伴侣蛋白 线粒体蛋白质组。然而，这些事件是如何在线粒体内分布和调控的 他们的动态仍然没有被很好地理解。此外，这些事件的时间安排是如何机械地 调节以维持适当的线粒体功能和整体细胞动态平衡仍不清楚。vbl.使用 先进的高速超分辨率显微镜和细胞器内的单分子跟踪研究，这 一项提案旨在研究细胞器内成分和单个蛋白质的动力学 复合体在线粒体等细胞器内受到调节。此外，由于线粒体DNA和 线粒体伴侣与多种人类疾病有关，这些疾病涉及神经退化， 这些研究还将阐明细胞器内事件是如何在神经元中优先调节的 在不同的神经元隔间和不同的神经元群体中调节神经元的稳态。 最后，这项工作将探讨细胞器内事件在驱动疾病发病机制中的作用。 衍生神经元为线粒体DNA和线粒体伴侣突变如何参与 神经疾病的病因，并有可能确定新的靶向治疗角度 神经退行性变。总之，这些研究将有助于回答关键组件如何 线粒体内的蛋白质组合在空间和时间上被组织起来，以动态调节 细胞和神经元的动态平衡。最终，这项工作将为研究内部机制提供一个新的平台。 在其他类型的细胞器中发生的细胞器事件，最终目标是创建一个动态的细胞 整个活细胞的架构图，扩展了我们对实时的基本理解 以细胞生物学和细胞神经科学为基础的纳米级分子事件。

PROJECT SUMMARY Cellular function is maintained by the intricate regulation and coordination of highly dynamic organelles, which themselves contain multiple components and protein assemblies that constantly function together to promote cellular homeostasis. However, the field of cell biology has thus far largely focused on organelle dynamics at the whole-organelle level, or the crosstalk between organelles at inter-organelle membrane contact sites. In contrast, the molecular events occurring inside organelles at the intra-organelle level, such as within the mitochondria matrix, and the timing, distribution and regulation of these events has remained vastly unexplored, largely due to the previous lack of high-speed super-resolution microscopy to probe these questions at the nanoscale level. Importantly, filling in these missing pieces will be essential for the field of cell biology by dynamically mapping the cellular architecture of a living cell in real-time and advancing our knowledge of cellular homeostasis at the intra-organelle level. Mitochondria are highly critical organelles for regulating cellular function, and contain their own mtDNA which is transcribed into mtRNA within the mitochondrial matrix, as well as mitochondrial chaperones which help to promote efficient folding of the mitochondrial proteome. However, how these events are distributed inside the mitochondria and the regulation of their dynamics is still not well understood. Moreover, how the timing of these events are mechanistically regulated to maintain proper mitochondrial function and overall cellular homeostasis remains unclear. Using advanced high-speed super-resolution microscopy and single molecule tracking studies inside organelles, this proposal seeks to investigate how the dynamics of intra-organelle components and individual protein complexes are regulated within organelles such as the mitochondria. In addition, as defects in mtDNA and mitochondrial chaperones have been linked to multiple human diseases which involve neurodegeneration, these studies will also shed light on how intra-organelle events are preferentially regulated in neurons to regulate neuronal homeostasis in distinct neuronal compartments and within different neuronal populations. Finally, this work will probe the role of intra-organelle events in driving disease pathogenesis using patient- derived neurons to provide new insights into how mtDNA and mitochondrial chaperone mutations contribute to the etiology of neurological disorders, and potentially identify new therapeutic angles for targeting neurodegeneration. Together, these studies will help to answer essential questions of how key components and protein assemblies inside the mitochondria are spatially and temporally organized to dynamically regulate cellular and neuronal homeostasis. Ultimately, this work will provide a novel platform for studying the intra- organelle events occurring within other types of organelles, with the end goal of creating a dynamic cellular architecture map of the entire living cell and expanding our fundamental understanding of the real-time molecular events at nanoscale levels underlying cell biology and cellular neuroscience.