BACKGROUND AND PURPOSE:Annexin A6 (AnxA6) is a calcium-dependent phospholipid binding protein that can be recruited to the plasma membrane to function as a scaffolding protein to regulate signal complex formation, endo- and exocytic pathways as well as distribution of cellular cholesterol. The purpose of this study was to investigate how AnxA6 influences the membrane order.EXPERIMENTAL APPROACH:We used Laurdan and di-4-ANEPPDHQ staining in [i] artificial membranes and [ii] live cells to investigate membrane packing and ordered lipid phases, and [iii] a super-resolution imaging (photoactivated localization microscopy, PALM) and Ripley's K second-order point-pattern analysis approach to assess how AnxA6 regulates... More
BACKGROUND AND PURPOSE:Annexin A6 (AnxA6) is a calcium-dependent phospholipid binding protein that can be recruited to the plasma membrane to function as a scaffolding protein to regulate signal complex formation, endo- and exocytic pathways as well as distribution of cellular cholesterol. The purpose of this study was to investigate how AnxA6 influences the membrane order.EXPERIMENTAL APPROACH:We used Laurdan and di-4-ANEPPDHQ staining in [i] artificial membranes and [ii] live cells to investigate membrane packing and ordered lipid phases, and [iii] a super-resolution imaging (photoactivated localization microscopy, PALM) and Ripley's K second-order point-pattern analysis approach to assess how AnxA6 regulates plasma membrane order domains and protein clustering.KEY RESULTS:In artificial membranes, purified AnxA6 induced a significant global increase in membrane order. However, confocal microscopy using di-4-ANEPPDHQ in live cells showed that cells expressing AnxA6, which reduces plasma membrane cholesterol levels and modifies the actin cytoskeleton meshwork, displayed a significant decrease in membrane order (∼15% and 30% in A431 and MEF cells, respectively). PALM data from Lck10 and Src15 membrane raft/non-raft markers revealed that AnxA6 expression induced clustering of both raft and non-raft markers. Altered clustering of Lck10 and Src15 in cells expressing AnxA6 was also observed after cholesterol extraction with methyl-β-cyclodextrin or actin cytoskeleton disruption with latrunculin B.CONCLUSIONS AND IMPLICATIONS:AnxA6-induced plasma membrane remodelling indicates that elevated AnxA6 expression significantly decreases membrane order through the regulation of cellular cholesterol homeostasis and the actin cytoskeleton. This study provides the first evidence from live cells that support current models of annexins as membrane organizers.
