PLD-dependent phosphatidic acid microdomains are signaling platforms for podosome formation

Abstract

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.

Modular actin nano-architecture enables podosome protrusion and mechanosensing

“…Another explanation is that other tension sensitive adaptor proteins regulate the activity or excretion of metalloproteases due to altered force distributions in podosome modules on soft substrates. We recently showed that on stiff substrates, MT1-MMP-dependent gelatin degradation is mediated by the action of phospholipase D 62 . It would be interesting to explore the role of this signaling pathway in podosome mechanosensing in future studies.…”

Phospholipase D2 restores endothelial barrier function by promoting PTPN14-mediated VE-cadherin dephosphorylation

“…Tyrosine phosphorylation of VEcadherin at 658 by c-Src, facilitates VE-cadherin internalization in a β-arrestin dependent manner (3), and the internalized VE-cadherin enters endosomal compartments and is either recycled to the plasma membrane or subjected to degradation (68). Recent studies have shown that spatiotemporally generated PA from PLD2 acts as signaling platforms for MT1-MMP surface trafficking and lung metastasis of breast cancer cells (69), ERK signaling in cancer cells (47), podosome formation (70), and recruitment of IQGAP1 associated with neo-intima formation (56). In addition to PLD2, PLD1 mediated PA generation in microdomains promotes vesicular fusion to the plasma membrane during exocytosis of large dense-core granules [70]; however, a role for PA confinement in microdomains in VE-cadherin redistribution to AJs is unknown.…”

Cytoskeletal regulation of dendritic cells: An intricate balance between migration and presentation for tumor therapy

“…In DCs, podosome formation is dependent on the recruitment of the Arp2/3 complex by WASp and their architecture is regulated by Cdc42, Rac, and Rho 107,135 . Using live cell imaging, recent work suggests that phosphatidic acid microdomains generated by phospholipase D drive podosome formation 136 . Podosomes are highly dynamic structures that exhibit rapid actin turnover rate as demonstrated by a fast reincorporation of GFP‐actin after photobleaching 137 .…”

“…107,135 Using live cell imaging, recent work suggests that phosphatidic acid microdomains generated by phospholipase D drive podosome formation. 136 Podosomes are highly dynamic structures that exhibit rapid actin turnover rate as demonstrated by a fast reincorporation of GFP-actin after photobleaching. 137 The exact mechanism of how podosomes are involved in DC migration is not yet identified.…”