Molecular organization of pathways governing cell-shape formation

控制细胞形状形成途径的分子组织

• 批准号: 10699748
• 负责人: Naoko Mizuno
• 金额: $ 193.95万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 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/10699748
• 关键词: Actins; Affect; Axon; Axotomy; Behavior; Binding; Biological; Cell Shape; Cell membrane; Cell physiology; Cells; Central Nervous System; 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; 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; base; biophysical techniques; brain cell; 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

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 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. Next, we aim to elucidate mechanisms underlying the maintenance and regeneration of neurons. We developed a robust 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 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.

光镜结合cryo-ET自顶向下细胞观察 为了便于细胞内成分的分子分析，我们已经建立了一个管道，用于制备原代小鼠神经元，并通过细胞冷冻电子断层扫描（cryo-ET）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，我们进行了超微结构分析的分支轴突从中枢神经系统（CNS）。作为第一次观察，我们已经获得了一些可视化的见解轴突形态发生和分支过程中的信号转导过程和细胞骨架重塑。通过冷冻ET对>100个轴突分支的系统研究，我们发现轴突分支点作为细胞动力学活动的热点，这在轴突轴的任何其他沿着位置都没有发现，促进了轴突内部更高水平的区室化。当一个分支仍然是新生的和过早的，它是可逆地形成通过形成肌动蛋白为基础的丝状伪足，但没有微管已经建立，以加强分支。在这个阶段，我们观察到线粒体和肌动蛋白丝的短片段的积累，使一个对齐的组织向丝状伪足的尖端。我们发现核糖体在胞浆中的共存，以及在ER专门在轴突分支点，这支持了局部蛋白质合成发生在轴突分支点，必要的新的轴突形成的假设。ER通常沿轴突轴沿着形成薄而光滑的结构，在轴突分支点膨胀成片状结构。它促进核糖体的结合，并作为分泌和膜蛋白的合成平台。轴突分支的成熟通过短微管进入分支而发生。在这里，我们观察到的共同迁移的ER和微管，这是必要的建立一个成熟的轴突分支。内质网膜缠绕在微管周围，使它们相互束缚以促进共迁移。我们获得了一个事件路线图，允许轴突分支站点作为轴突发育和下游神经网络形成的独特控制中心。 接下来，我们的目标是阐明神经元的维持和再生的机制。我们开发了一个强大的管道来模拟轴突损伤（轴突切断术），并测试了一系列可能是轴突再生的关键因素。我们已经确定了关键目标，并获得了初步结果，显示细胞成分的重组 自下而上重构细胞形状形成的关键信号复合物及其验证。 控制细胞和神经元形状形成的最重要的途径之一是粘着斑（FA）和FA相关的信号传导。FA是由其主受体整合素控制的细胞机器，整合素对细胞形状形成具有广泛的作用。FA包含数百个分子参与者，在质膜上产生多层蛋白质-蛋白质网络，最终连接到肌动蛋白细胞骨架以及细胞信号传导因子。由于调控层协调了系统的正常功能，因此在分子水平上阐明FA作为一个整体是不可能的，并且阻碍了我们对FA调控的理解。为了检验这一假设，即关键组分的分子功能是以分层的方式进行调节的，我们采用了自下而上的重建方法，旨在建立模拟FA启动的大分子机制。利用光学显微镜，冷冻电镜和生物物理学方法，我们已经阐明了FA，整合素，talin，黏着斑蛋白和肌动蛋白及其在膜表面的组装过程的主控制器的调节机制。我们目前正在使用cryo-EM研究依赖于Rap 1信号传导的整合素激活机制。Rap 1介导FA通路与其他细胞/信号通路之间的串扰。我们正在分析Rap 1在转换过程中的功能相关性。使用上述自上而下的方法在细胞背景下测试从体外重建中吸取的经验教训。