Annexin A2 modulates phospholipid membrane composition upstream of Arp2 to control angiogenic sprout initiation

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.

Opportunistic pathogen Porphyromonas gingivalis targets the LC3B-ceramide complex and mediates lethal mitophagy resistance in oral tumors

“…Among the multiple LC3B-associated proteins identified ( Figure S3 A), we focused our attention on Annexin A2 (ANXA2) due to its known roles in calcium-dependent phospholipid-binding, participation in endocytosis/exocytosis, and activity linking F-actin cytoskeleton to the plasma membrane. 40 , 41 , 42 We validated LC3B-ANXA2 association by co-IP and IF using anti-LC3B and anti-ANXA2 antibodies ( Figures 2 A and 2B). Interestingly, these results showed that P. gingivalis infection blunted LC3B-associated ANXA2 levels ∼75–80% with or without LCL768 in UM-SCC-1A cells ( Figures 2 A and 2B) without any changes in overall intracellular protein levels of ANXA2 ( Figure 2 A, input panel).…”

“…However, it remains unknown whether ANXA2 is involved in ceramide trafficking from the inner to outer leaflet of the mitochondrial membrane, or from the endoplasmic reticulum (ER) to mitochondria. It has been previously reported that ANXA2 is able to bind and extract lipids from membranes, 42 , 57 , 58 , 59 , 60 which suggest that ANXA2 might also be able to induce ceramide trafficking from ER to mitochondria, then triggering initial mitochondrial ceramide signaling. Mitochondrial accumulation of ceramide is known to induce the generation of reactive oxygen species that in turn activates Drp1 and CerS1-p17/PERMIT trafficking to induce hyper-mitophagy, 26 which then triggers lethal mitophagy.…”