质膜下肌动蛋白聚合触发囊泡-质膜半融合（hemifusion）形成的机制研究

The plasma membrane and most organelles of eukaryotic cells have a biofilm structure composed of lipid bilayers. Fusion between membrane structures is an important and fundamental biological process. Hemi-fusion (ie, the state of pre-fusion of the contacted lipid monolayers of the two membrane structures) is an important intermediate structure in the process of membrane fusion and the mechanism of its regulation is unknown. The applicant have innovatively constructed an experimental system for analyzing the hemi-fusion between the secreted vesicle and the plasma membrane in live cells using microscopic real-time imaging technique in conjunction with whole-cell patch clamp technique (Zhao WD et al. Nature. 2016). The applicant recently found for the first time that actin, an important component of the cytoskeleton, was involved in the regulation of vesicle and plasma membrane hemi-fusion formation. Therefore, this project intends to use conditional knock-out transgenic mice with different isoforms of actin to further analyze the role of actin in the formation of vesicle hemi-fusion, identify key molecules that trigger actin polymerization, clarify the regulatory effect of actin on the hemi-fusion "checkpoint", and then illuminate the role of actin in vesicle hemi-fusion and its mechanism. This project will reveal the role of actin in membrane hemi-fusion, propose a new actin-regulated hemi-fusion signaling pathway and promote the understanding of the mechanism of membrane fusion in eukaryotic cells.

真核细胞的质膜和多数细胞器都具备由脂质双层构成的生物膜结构。膜结构之间的融合是重要而基础的生物学过程。半融合（即两个膜结构的相接触的脂质单层先行融合的状态）是膜融合过程重要的中间结构，然而其调控机制不明。申请者利用显微实时成像联合全细胞膜片钳技术，创新性地构建了在活细胞中解析分泌囊泡与质膜间的半融合的实验体系（Zhao WD et al. Nature. 2016）。申请者最近首次发现，细胞骨架的重要组分——肌动蛋白，参与调控囊泡-质膜的半融合形成。鉴此，本项目拟利用条件性敲除不同亚型肌动蛋白的转基因小鼠，深入解析肌动蛋白在囊泡半融合形成中的作用，鉴定半融合时触发肌动蛋白聚合的关键分子，进一步明确肌动蛋白对半融合“检查点”功能的调控作用，从而揭示肌动蛋白在囊泡半融合形成中的作用和机理。本项目将提出新的肌动蛋白调控半融合形成的信号通路，推动对真核细胞中膜融合机理的认识。

肌动蛋白作为细胞骨架的重要组分，在细胞的囊泡融合及释放中的作用机制及其生物学意义目前尚不清楚。本项目利用单细胞测序、条件性敲除不同亚型肌动蛋白的转基因小鼠等技术，深入解析了肌动蛋白在囊泡融合及释放中的作用，并进一步而揭示肌动蛋白所调控的囊泡形成的生物学意义。目前的主要研究进展为：（1）发现肌动蛋白亚型能够调控囊泡的释放，且周细胞释放的囊泡能够与邻近的脑微血管内皮细胞发生融合，促进脑微血管的血管新生能力；（2）建立了对不同亚型的肌动蛋白进行单独及联合敲降的体内和体外实验体系，发现肌动蛋白亚型之间的功能存在相互代偿，进一步发现联合敲降肌动蛋白亚型抑制血管发育过程及尖端细胞依赖性血管新生；（3）发现了肌动蛋白亚型的新功能，即能够维持脑微血管内皮细胞分化状态的稳定，联合敲降肌动蛋白亚型诱发血管内皮细胞向间质方向转化；（4）建立了在活细胞中对肌动蛋白亚型进行标记的技术体系（中国医科大学学报, 2020, 49(8): 673-678）；（5）发现肌动蛋白亚型参与巨噬细胞对脑膜炎大肠杆菌的吞噬过程（中国细胞生物学学报, 2020, 42: 620-628）；（6）在对免疫细胞吞噬脑膜炎大肠杆菌的研究中发现，细菌Lpp脂蛋白能够抑制氧自由基生成从而避免中性粒细胞的杀伤（Redox Biology, 2023, 59: 102588）。