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Molecular organization of pathways governing cell-shape formation

控制细胞形状形成途径的分子组织

基本信息

批准号：
10929201
负责人：
Naoko Mizuno
金额：
$ 214.75万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10929201
关键词：
Actins Affect Axon Axotomy Behavior Binding Biochemical Biological Brain Cell Shape Cell membrane Cell physiology Cells Central Nervous System Communication Complex Cryo-electron tomography Cryoelectron Microscopy Cytoskeleton Cytosol Decision Making Deterioration Development Distal Elements Endoplasmic Reticulum Environment Event Filopodia Focal Adhesions Fostering In Situ In Vitro Individual Integrins Learning Length Light Microscope Maintenance Mediating Membrane Membrane Proteins Methods Microfilaments Microtubules Mitochondria Molecular Molecular Analysis Morphogenesis Mus Natural regeneration Neoplasm Metastasis Nerve Degeneration Nervous System Neurofibrillary Tangles Neurons Organelles Pathologic Pathway interactions Play Preparation Process Protein Biosynthesis Proteins Regulation Ribosomes Role Series Shapes Signal Pathway Signal Transduction Site Structure Surface Surveys System Talin Testing Thinness Time Validation Vinculin Visual axon injury axon regeneration biophysical techniques cell motility insight interest light microscopy macromolecular assembly migration neural network neuron regeneration polarized cell premature receptor reconstitution repaired response secretory protein stem tumor growth

项目摘要

Top-down cellular observation using light microscope combined with cryo-ET To facilitate the molecular analysis of intracellular components, we have established a pipeline for the preparation of primary mouse neurons, and their observation in situ by cellular cryo-electron tomography (cryo-ET). The shape formation of neurons and the role of cellular signaling activities at axon branching points is of particular interest. Therefore, we have conducted ultrastructural analyses of branching axons from the central nervous system (CNS). As the first time observation, we have obtained a number of visual insights into signaling processes and cytoskeleton remodeling during axon morphogenesis and branching. Through a systematic survey of >100 axon branches by cryo-ET, we found that axon branching points act as hotspots for cellular dynamic activities, which is not found at any other places along the axon shaft, fostering a higher level of compartmentalization inside axons. When a branch is still nascent and premature, it is reversibly formed via a formation of actin-based filopodia but no microtubules have been established to reinforce the branch. At this stage, we observed an accumulation of mitochondria and short fragments of actin filaments, making an aligned organization towards the tip of filopodia. We discovered the coexistence of ribosomes in cytosol, as well as on ER exclusively at the axon branching point, which supports the hypothesis of local protein synthesis occurring at axon branch points, necessary for the new axon formation. The ER, which normally forms a thin, smooth formation along the axon shaft, expands at the axon branch point into a sheet-like formation. It fosters the binding of ribosomes and acts as a synthesis platform for secretory and membrane proteins. Maturation of the axon branch occurs by the entering of short microtubules into the branch. Here, we observed the co-migration of ER and microtubules, which is necessary for establishing a matured axon branch. ER membranes were tangled around the microtubules so that they tether each other to facilitate the comigration. We obtained a roadmap of events allowing axon branching sites to serve as unique control hubs for axon development and downstream neural network formation. After this showcase study, we aim to elucidate mechanisms underlying the maintenance and regeneration of neurons. We developed a pipeline to mimic axon injury (axotomy) and tested a series of factors that might be key for axon regeneration. We have identified key targets and obtained preliminary results showing the reorganization of cellular components. Bottom-up reconstitution of key signaling complexes for cell shape formation and their validation. One of the most important pathways that is controlling the cellular and neuronal shape formation is focal adhesion (FA) and FA-related signaling. FA is a cellular machinery controlled by its master receptor, integrin, which has wide-ranging roles for cell shape formation. FA contains a few hundred molecular players generating a multi-layered protein-protein network at the plasma membrane, which ultimately connects to the actin cytoskeleton as well as cellular signaling factors. As layers of regulations orchestrate the proper functioning of the system, elucidating FA as a whole on a molecular level is not attainable and hindering our understanding of the FA regulation. To test the hypothesis that the molecular functions of key components are regulated in a hierarchical fashion, we employ a bottom-up reconstitution approach and aim to build up the macromolecular machinery that would mimic FA initiation. Using light microscopy, cryo-EM and biophysical methods, we have elucidated the mechanisms of regulation of the master controllers of FA, integrin, talin, vinculin and actin and their assembly process at the membrane surface. We analyze the downstream of the FA activation, by obtaining structural insights into actin-talin-vinculin interactions by using cryo-EM approaches in combination with biochemical interaction analyses. Furthermore, we are currently working on the integrin activation mechanisms that rely on Rap1 signaling using cryo-EM. Rap1 mediates the crosstalk between the FA pathway and other cellular/signaling pathways. We are analyzing the functional relevance of Rap1 in the switching process. Lessons learned from the in vitro reconstitution are tested in a cellular context using the top-down approach mentioned above.

光学显微镜结合低温电子显微镜自上而下观察细胞为了便于细胞内成分的分子分析，我们建立了一条制备原代小鼠神经元的管道，并用细胞冷冻电子断层扫描(Cryo-ET)对其进行了原位观察。神经元的形状形成和轴突分支点的细胞信号活动的作用特别令人感兴趣。因此，我们对中枢神经系统(CNS)的分支轴突进行了超微结构分析。作为第一次观察，我们获得了许多关于轴突形态发生和分支过程中的信号过程和细胞骨架重塑的可视化见解。通过低温对100个轴突分支的系统调查，我们发现轴突分支点是细胞动力学活动的热点，这是轴突干上任何其他地方都没有的，促进了轴突内部更高水平的区域化。当一个分支仍然是新生的和早熟的，它是通过形成基于肌动蛋白的丝状伪足可逆地形成的，但是没有微管被建立来加强分支。在这个阶段，我们观察到线粒体和肌动蛋白细丝的短片段的积累，使组织朝着丝状足端排列。我们发现核糖体在胞浆中共存，并且仅在轴突分叉点的内质网上共存，这支持了局部蛋白质合成发生在轴突分支点的假说，这是新轴突形成所必需的。内质网通常沿着轴突形成一个薄而光滑的结构，但在轴突分支点扩张为片状结构。它促进核糖体的结合，并作为分泌和膜蛋白的合成平台。轴突分支的成熟是通过短的微管进入分支而发生的。在这里，我们观察到内质网和微管的共同迁移，这是建立成熟的轴突分支所必需的。ER膜缠绕在微管周围，使它们相互捆绑在一起，促进移行。我们得到了一个事件路线图，允许轴突分支位置作为轴突发育和下游神经网络形成的独特控制中心。在这项示范研究之后，我们的目标是阐明神经元维持和再生的潜在机制。我们开发了一种模拟轴突损伤(轴突切断)的管道，并测试了一系列可能对轴突再生至关重要的因素。我们已经确定了关键靶点，并获得了显示细胞成分重组的初步结果。用于细胞形状形成的关键信号复合体的自下而上的重构及其有效性。控制细胞和神经元形态形成的最重要的途径之一是焦点黏附(FA)及其相关信号。FA是一种受其主要受体整合素控制的细胞器，整合素在细胞形态形成中具有广泛的作用。FA包含数百个分子玩家，在质膜上产生一个多层的蛋白质-蛋白质网络，最终连接到肌动蛋白细胞骨架和细胞信号因子。由于层层的法规协调着系统的正常运作，在分子水平上阐明FA作为一个整体是不可能的，并阻碍了我们对FA法规的理解。为了验证关键成分的分子功能以分级方式调节的假设，我们采用了自下而上的重组方法，旨在建立模拟FA启动的大分子机制。利用光学显微镜、低温电子显微镜和生物物理方法，我们阐明了FA、整合素、talin、vinculin和actin的主控制器的调控机制及其在膜表面的组装过程。我们通过使用冷冻-EM方法结合生化相互作用分析来获得对肌动蛋白-talin-vinculin相互作用的结构洞察力，从而分析FA激活的下游。此外，我们目前正在研究整合素激活机制，该机制依赖于使用Cryo-EM的Rap1信号。RAP1介导FA通路和其他细胞/信号通路之间的串扰。我们正在分析Rap1在交换过程中的功能相关性。从体外重建中学到的经验教训是在细胞环境中使用上述自上而下的方法进行测试的。