Fluorescent Annexin A1 Reveals Dynamics of Ceramide Platforms in Living Cells

Abstract

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 21 , 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 21 concentrations, this leads to an increase in the Ca 21 sensitivity of annexin A1-membrane interaction. In fixed cells, using a ceramidespecific 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 21 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.

Intracellular Ca2+ operates a switch between repair and lysis of streptolysin O-perforated cells

“…Recently, we have established the formation and internalisation of annexin A1-positive platforms to be the result of the self-association of ceramide, generated by a Ca 2 þ -dependent activation of acid sphingomyelinase. 12 The co-localisation of annexin A1 with ceramide within the platforms was confirmed using a ceramide-specific antibody. 12 Annexin A1 and -to a much lesser degree -annexin A6 were also localised in the nucleus (Figure 2).…”

“…12 The co-localisation of annexin A1 with ceramide within the platforms was confirmed using a ceramide-specific antibody. 12 Annexin A1 and -to a much lesser degree -annexin A6 were also localised in the nucleus (Figure 2). The translocation of both annexins to the plasma membrane preceded their translocation from an intranuclear compartment to the nuclear envelope most likely due to the relative abundance of negatively charged phospholipids within the plasma membrane.…”

“…Immediately after its translocation, annexin A1 was evenly distributed at the plasma membrane, co-localising with annexin A6 (Figure 2c; 54 s). However, within seconds, annexin A1 segregated into lateral plasma membrane platforms, which were subsequently internalised 12 (Figure 2c; 72 s). Recently, we have established the formation and internalisation of annexin A1-positive platforms to be the result of the self-association of ceramide, generated by a Ca 2 þ -dependent activation of acid sphingomyelinase.…”

Blebbing confers resistance against cell lysis

“…A Ca 2 þ -induced formation of ceramide within the plasma membrane exposed to the Ca 2 þ hot-spot might further increase the selectivity of annexin A1 binding. 32 Thus concentrated, annexin A1 cross-links adjacent membranes of a slender neck of the bleb, thereby forming a plug. An efflux of cytosol is thus prevented and extracellular 'invaders' remain trapped within the injured bleb.…”

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

“…Annexins are a family of structurally related Ca 2+ -regulated membrane-binding proteins. Among the 12 members of the mammalian family, annexins (Anx) A1, A2, A6, A8, and A13b have been particularly implicated in a variety of intracellular trafficking steps along endo- and exocytic pathways (Fiedler et al , 1995; Mayran et al , 2003; Babiychuk et al , 2008; Goebeler et al , 2008; Grewal and Enrich, 2009; Monastyrskaya et al , 2009a; Morel et al , 2009). …”