基于超分辨成像技术对嘌呤受体介导的小胶质细胞微丝骨架重构的研究

Microglia acts as the first immune defense in the central nervous system (CNS). During brain injury or infection, diverse nucleotides release or leak from apoptotic neurons, some of which act as “find/eat-me” signals. By activating cell-surface purinergic receptors and inducing cell signal transduction, it leads to oriented migration of microglia toward injured sites and subsequently promotes phagocytic clearance of dead cells and tissue debris by microglia, which contributes to maintain homeostasis of the CNS. The rearrangement of cytoskeleton network is essential for this directed migration process. However, previous studies on microglial cytoskeleton were largely insufficient due to diffraction limit in optical microscopy field. Diversity of super-resolution fluorescence microscopy technologies that were developed in recent years overcame the diffraction limit and improved imaging resolution to less than hundred nanometers, which greatly enriched the investigation of sub-cellular structures and function. Therefore, in the present project, we intend to explore the fine structure of microfilament cytoskeleton network in microglia based on stochastic optical reconstruction microscopy (STORM), and observe the dynamic assembly of characteristic structure such as lamellipodium and podosome during the process of directional migration by structured illumination microscopy (SIM). Moreover, we will clarify the role of purine receptors and related calcium signaling in cytoskeletal reconstruction. This project will contribute to further understanding of cytoskeleton reconstruction during microglia migration, and reveal the regulatory mechanism in this process, thus give better knowledge of the protection mechanism by microglia in the CNS and novel insight in regulating cell migration by controlling cytoskeleton remodeling.

小胶质细胞是中枢神经系统的第一道免疫防线。当脑部出现损伤或感染时，会产生大量核苷酸信号分子，通过活化细胞膜上的P2类嘌呤受体启动细胞信号转导，使小胶质细胞迁移至病灶区，清除神经碎片和病变组织，保障神经系统的正常运行。细胞骨架体系的重构为这一定向移动过程提供主要动力。然而，受限于光学衍射极限，以往对于小胶质细胞骨架的研究存在很大不足。本项目拟基于近年发展起来的STORM和SIM超分辨成像技术解析不同条件下小胶质细胞微丝骨架网络的精细结构，及实时观测核苷酸诱发细胞趋化性迁移时片状伪足、伪足小体等结构的动态组装和转变过程。进一步，探究嘌呤受体及其介导的钙信号通路在细胞微丝骨架重构中的作用，确定其中关键的信号环节。本研究有助于深入了解小胶质细胞定向迁移时骨架重构的过程和调节机制，从而更好地理解和认识小胶质细胞保护中枢神经系统的机理，并为实现以细胞骨架为靶点调控细胞迁移提供新的研究思路。

小胶质细胞是中枢神经系统第一道免疫防线。在神经损伤和感染过程中，其通过定向迁移至病灶部位发挥免疫作用。本项目以新生乳鼠大脑皮层原代小胶质细胞为研究对象，基于超分辨成像技术，着眼于细胞微丝骨架的重构和细胞膜嘌呤受体的变化，探究小胶质细胞迁移过程的微观机制。首先通过3D-STORM超分辨成像和活细胞微丝骨架成像揭示了迁移态小胶质细胞片状伪足区域“玫瑰花环”伪足小体超结构的形成和动态组装-解聚过程，阐明了细胞肌动球蛋白和微管骨架网络对伪足小体超结构形成和维持的调控作用。并解析了小胶质细胞伪足小体纳米尺度的精细结构，获得了普通荧光成像无法定量表征的伪足小体半径、高度和分布密度等参数。另一方面，揭示了迁移相关嘌呤受体P2Y12在小胶质细胞膜上的空间组织和分布特性，发现静息态细胞中P2Y12受体随机分布于细胞膜，而迁移态细胞中P2Y12受体显著富集于细胞前导端局部位置，通过STORM图像分析证明部分受体还定位于伪足小体肌动蛋白核心周围的环结构上。P2Y12膜受体的空间组织特性也受到细胞微丝、微管骨架网络的调制。而且，P2Y12受体及其介导的下游Ca2+/SOCE/CaMKII信号通路在调控小胶质细胞玫瑰花环伪足小体超结构中发挥关键作用。进一步，以胞外嘌呤类核苷酸ADP作为趋化因子，利用微注射趋化系统建立稳定、可控的趋化浓度梯度诱导小胶质细胞定向迁移，发现细胞在迁移过程中会发生极化并在前导端形成“玫瑰花环”伪足小体超结构，P2Y12受体局域化分布于细胞膜前导端特定部位，并朝向趋化因子浓度梯度，明确了该受体在感受趋化因子和指导细胞迁移方向中的重要作用。相关结果有助于阐明小胶质细胞迁移时微丝骨架重构的过程和调节机制，从而为深入认识小胶质细胞保护中枢神经系统的机理和实现以细胞骨架为靶点调控细胞迁移能力提供新的实验支撑和新的视角。