CD2AP介导的液-液相分离调控小胶质细胞的吞噬和迁移

CD2AP-mediated liquiol-liquid phase separation regulates microglia phagocytosis and migration

摘要:

观察 CD2AP 在小鼠大脑中的表达情况，探究其是否会发生相分离，以及对小胶质细胞吞噬和 迁移功能的影响。方法 使用免疫荧光染色法研究CD2AP蛋白在WT小鼠和 AD 模型小鼠大脑中的表达和定位 情况；在转染 CD2AP 的 293T 细胞中检测液滴的液态动力学；使用 CD2AP 重组蛋白观察液滴的形成；体外培养 HMC3 细胞并转染，通过划痕实验和细胞吞噬实验，检测对照组和 CD2AP 组细胞迁移率和吞噬的差异。结果 CD2AP 在小胶质细胞中呈高表达，呈液滴状分布。胞内过表达CD2AP后出现了具有流动性的液滴。CD2AP 体外 重组蛋白在不同拥挤剂的作用下不同时间点均能形成液滴。CD2AP 过表达组细胞迁移率明显增高（P < 0.05），但 吞噬能力下降（P < 0.05）。结论 CD2AP 具有液-液相分离的能力，并且影响小胶质细胞的吞噬和迁移能力。

Abstract:

Objective To observe the expression of CD2AP in the mouse brain, investigate whether it undergoes phase separation, and assess its effect on the phagocytic and migratory functions of microglia. Methods Immunofluorescence staining was used to study the expression and localization of CD2AP protein in the brains of wild-type (WT) mice and Alzheimer's disease (AD) model mice. The liquid dynamics of droplets was detected in 293T cells transfected with CD2AP. The formation of droplets was observed using CD2AP recombinant protein. HMC3 cells were cultured and transfected in vitro, and the differences in cell mobility and phagocytosis between control and CD2AP groups were assessed using scratch assay and cell phagocytosis assay. Results CD2AP was highly expressed in microglia, exhibiting a droplet-like distribution. Intracellular overexpression of CD2AP resulted in droplets exhibiting fluidity. In vitro, recombinant protein CD2AP formed droplets at various time points in the presence of different crowding agents. Cell scratch assay and phagocytosis assays showed that the CD2AP overexpression group had significantly higher cell migration (P<0.05) but decreased phagocytosis (P<0.05). Conclusion CD2AP has the ability to undergo liquid-liquid phase separation and affects the phagocytic and migratory abilities of microglia.