title: Quantitative analysis of self-association and mobility of annexin A4 at the plasma membrane.
abstract: Annexins, found in most eukaryotic species, are cytosolic proteins that are able to bind negatively-charged phospholipids in a calcium-dependent manner. Annexin A4 (AnxA4) has been implicated in diverse cellular processes, including the regulation of exocytosis and ion-transport; however, its precise mechanistic role is not fully understood. AnxA4 has been shown to aggregate on lipid layers upon Ca(2+) binding in vitro, a characteristic that may be critical for its function. We have utilized advanced fluorescence microscopy to discern details on the mobility and self-assembly of AnxA4 after Ca(2+) influx at the plasma membrane in living cells. Total internal reflection microscopy in combination with Förster resonance energy transfer reveals that there is a delay between initial plasma membrane binding and the beginning of self-assembly and this process continues after the cytoplasmic pool has completely relocated. Number-and-brightness analysis suggests that the predominant membrane bound mobile form of the protein is trimeric. There also exists a pool of AnxA4 that forms highly immobile aggregates at the membrane. Fluorescence recovery after photobleaching suggests that the relative proportion of these two forms varies and is correlated with membrane morphology.
authors.affiliation: Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, Netherlands.
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Crosby
authors.forename: Marten
authors.initials: M
authors.lastname: Postma
authors.forename: Mark A
authors.initials: MA
authors.lastname: Hink
authors.forename: Christiaan H C
authors.initials: CH
authors.lastname: Zeelenberg
authors.forename: Merel J W
authors.initials: MJ
authors.lastname: Adjobo-Hermans
authors.forename: Theodorus W J
authors.initials: TW
authors.lastname: Gadella
mesh_terms: D017314:Annexin A4 / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006801:Humans; D055503:Protein Multimerization
chemical_list: D017314:Annexin A4; D002118:Calcium