Fluorescent annexin A1 reveals dynamics of ceramide platforms in living cells.
Upon its genesis during apoptosis, ceramide promotes gross reorganization of the plasma membrane structure involving clustering of signalling molecules and an amplification of vesicle formation, fusion and trafficking. The annexins are a family of proteins, which in the presence of Ca(2+), bind to membranes containing negatively charged phospholipids. Here, we show that ceramide increases affinity of annexin A1-membrane interaction. In the physiologically relevant range of Ca(2+) concentrations, this leads to an increase in the Ca(2+)sensitivity of annexin A1-membrane interaction. In fixed cells, using a ceramide-specific antibody, we establish a direct interaction of annexin A1 with areas of the plasma membrane enriched in ceramide (ceramide platforms). In living cells, the intracellular dynamics of annexin A1 match those of plasmalemmal ceramide. Among proteins of the annexin family, the interaction with ceramide platforms is restricted to annexin A1 and is conveyed by its unique N-terminal domain. We demonstrate that intracellular Ca(2+)overload occurring at the conditions of cellular stress induces ceramide production. Using fluorescently tagged annexin A1 as a reporter for ceramide platforms and annexin A6 as a non-selective membrane marker, we visualize ceramide platforms for the first time in living cells and provide evidence for a ceramide-driven segregation and internalization of membrane-associated proteins.
Babiychuk
Eduard B
EB
Department of Cell Biology, Institute of Anatomy, University of Bern, Bern, Switzerland. edik@ana.unibe.ch
Monastyrskaya
Katia
K
Draeger
Annette
A
D000818:Animals; D017305:Annexin A1 / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002104:Cadmium / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D002518:Ceramides / Q000378:metabolism / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D018613:Microscopy, Confocal; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D013108:Sphingomyelin Phosphodiesterase / Q000037:antagonists & inhibitors / Q000378:metabolism; D013552:Swine; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism
D017305:Annexin A1; D002518:Ceramides; D002104:Cadmium; D015759:Ionomycin; D017855:Transferases (Other Substituted Phosphate Groups); C070186:phosphatidylcholine-ceramide phosphocholine transferase; D013108:Sphingomyelin Phosphodiesterase; D002118:Calcium