Department of Cell Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
Microscopic Imaging Center, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands.
Sci Rep. 2019 Mar 5;9(1):3556. doi: 10.1038/s41598-019-39358-0.
Local membrane phospholipid enrichment serves as docking platform for signaling proteins involved in many processes including cell adhesion and migration. Tissue-resident dendritic cells (DCs) assemble actomyosin-based structures called podosomes, which mediate adhesion and degradation of extracellular matrix for migration and antigen sampling. Recent evidence suggested the involvement of phospholipase D (PLD) and its product phosphatidic acid (PA) in podosome formation, but the spatiotemporal control of this process is poorly characterized. Here we determined the role of PLD1 and PLD2 isoforms in regulating podosome formation and dynamics in human primary DCs by combining PLD pharmacological inhibition with a fluorescent PA sensor and fluorescence microscopy. We found that ongoing PLD2 activity is required for the maintenance of podosomes, whereas both PLD1 and PLD2 control the early stages of podosome assembly. Furthermore, we captured the formation of PA microdomains accumulating at the membrane cytoplasmic leaflet of living DCs, in dynamic coordination with nascent podosome actin cores. Finally, we show that both PLD1 and PLD2 activity are important for podosome-mediated matrix degradation. Our results provide novel insight into the isoform-specific spatiotemporal regulation of PLD activity and further our understanding of the role of cell membrane phospholipids in controlling localized actin polymerization and cell protrusion.
局部膜磷脂富集可作为参与多种过程（包括细胞黏附和迁移）的信号蛋白的对接平台。组织驻留树突状细胞（DC）组装肌动球蛋白为基础的结构，称为足突，其介导细胞外基质的黏附和降解以进行迁移和抗原采样。最近的证据表明，磷脂酶 D（PLD）及其产物磷酸脂酸（PA）参与了足突的形成，但该过程的时空调控特征描述不足。在这里，我们通过结合 PLD 药理学抑制、荧光 PA 传感器和荧光显微镜，确定了 PLD1 和 PLD2 同工型在调节人原代 DC 中足突形成和动力学中的作用。我们发现，持续的 PLD2 活性是维持足突所必需的，而 PLD1 和 PLD2 均控制着足突组装的早期阶段。此外，我们还捕获了 PA 微区在活 DC 质膜胞质小叶的形成，与新生足突肌动蛋白核心动态协调。最后，我们表明 PLD1 和 PLD2 的活性对于足突介导的基质降解都很重要。我们的研究结果为 PLD 活性的同工型特异性时空调控提供了新的见解，并进一步加深了我们对细胞膜磷脂在控制局部肌动蛋白聚合和细胞突起中的作用的理解。
