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Abstract：
The intersection of protein and lipid biology is of growing importance for understanding how cells address structural challenges during adhesion and migration. While protein complexes engaged with the cytoskeleton play a vital role, support from the phospholipid membrane is crucial for directing localization and assembly of key protein complexes. During angiogenesis, dramatic cellular remodeling is necessary for endothelial cells to shift from a stable monolayer to invasive structures. However, the molecular dynamics between lipids and proteins during endothelial invasion are not defined. Here, we utilized cell culture, immunofluorescence, and lipidomic analyses to identify a novel role for the membrane binding protein Annexin A2 (ANXA2) in modulating the composition of specific membrane lipids necessary for cortical F-actin organization and adherens junction stabilization. In the absence of ANXA2, there is disorganized cortical F-actin, reduced junctional Arp2, excess sprout initiation, and ultimately failed sprout maturation. Furthermore, we observed reduced filipin III labeling of membrane cholesterol in cells with reduced ANXA2, suggesting there is an alteration in phospholipid membrane dynamics. Lipidomic analyses revealed that 42 lipid species were altered with loss of ANXA2, including an accumulation of phosphatidylcholine (16:0_16:0). We found that supplementation of phosphatidylcholine (16:0_16:0) in wild-type endothelial cells mimicked the ANXA2 knock-down phenotype, indicating that ANXA2 regulated the phospholipid membrane upstream of Arp2 recruitment and organization of cortical F-actin. Altogether, these data indicate a novel role for ANXA2 in coordinating events at endothelial junctions needed to initiate sprouting and show that proper lipid modulation is a critical component of these events.
摘要：
蛋白质和脂质生物学的交叉对于理解细胞如何解决粘附和迁移过程中的结构挑战越来越重要。虽然与细胞骨架有关的蛋白质复合物起着至关重要的作用，磷脂膜的支持对于指导关键蛋白质复合物的定位和组装至关重要。在血管生成过程中,急剧的细胞重塑对于内皮细胞从稳定的单层结构转变为侵入性结构是必要的。然而,在内皮侵入过程中，脂质和蛋白质之间的分子动力学尚不明确。这里,我们利用细胞培养，免疫荧光，和脂质组学分析，以确定膜结合蛋白膜联蛋白A2（ANXA2）在调节皮质F-肌动蛋白组织和粘附连接稳定所必需的特定膜脂质组成中的新作用。在没有ANXA2的情况下，有混乱的皮质F-肌动蛋白，减少的连接Arp2，过量的发芽，最终未能发芽成熟。此外，我们在ANXA2减少的细胞中观察到膜胆固醇的filipinIII标记减少，这表明磷脂膜动力学发生了改变。脂质组学分析揭示了42种脂质种类随着ANXA2的丧失而改变,包括磷脂酰胆碱(16:0_16:0)的积累。我们发现，在野生型内皮细胞中补充磷脂酰胆碱（16：0_16：0）模仿ANXA2敲低表型，表明ANXA2调节Arp2募集上游的磷脂膜和皮质F-肌动蛋白的组织。总之,这些数据表明ANXA2在协调启动发芽所需的内皮连接处事件中的新作用,并表明适当的脂质调节是这些事件的关键组成部分.
