title: Evidence for annexin A6-dependent plasma membrane remodelling of lipid domains.
abstract: OBJECTIVE
  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. Here, we have investigated how AnxA6 influences the membrane order.
  
  
  METHODS
  We used Laurdan and di-4-ANEPPDHQ staining in (i) artificial membranes; (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.
  
  
  RESULTS
  In artificial membranes, purified AnxA6 induced a 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 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
  AnxA6-induced plasma membrane remodelling indicated that elevated AnxA6 expression decreased 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.
authors.affiliation: Departament de Biologia Cel·lular, Immunologia i Neurociències, Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain.
authors.forename: Anna
authors.initials: A
authors.lastname: Alvarez-Guaita
authors.forename: Sandra
authors.initials: S
authors.lastname: Vilà de Muga
authors.forename: Dylan M
authors.initials: DM
authors.lastname: Owen
authors.forename: David
authors.initials: D
authors.lastname: Williamson
authors.forename: Astrid
authors.initials: A
authors.lastname: Magenau
authors.forename: Ana
authors.initials: A
authors.lastname: García-Melero
authors.forename: Meritxell
authors.initials: M
authors.lastname: Reverter
authors.forename: Monira
authors.initials: M
authors.lastname: Hoque
authors.forename: Rose
authors.initials: R
authors.lastname: Cairns
authors.forename: Rhea
authors.initials: R
authors.lastname: Cornely
authors.forename: Francesc
authors.initials: F
authors.lastname: Tebar
authors.forename: Thomas
authors.initials: T
authors.lastname: Grewal
authors.forename: Katharina
authors.initials: K
authors.lastname: Gaus
authors.forename: Jesús
authors.initials: J
authors.lastname: Ayala-Sanmartín
authors.forename: Carlos
authors.initials: C
authors.lastname: Enrich
authors.forename: Carles
authors.initials: C
authors.lastname: Rentero
mesh_terms: D000818:Animals; D017317:Annexin A6 / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008055:Lipids / Q000737:chemistry; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence
chemical_list: D017317:Annexin A6; D008055:Lipids