The targeting of plasmalemmal ceramide to mitochondria during apoptosis

Ceramide is a key lipid mediator of cellular processes such as differentiation, proliferation, growth arrest and apoptosis. During apoptosis, ceramide is produced within the plasma membrane. Although recent data suggest that the generation of intracellular ceramide increases mitochondrial permeability, the source of mitochondrial ceramide remains unknown. Here, we determine whether a stress-mediated plasmalemmal pool of ceramide might become available to the mitochondria of apoptotic cells. We have previously established annexin A1--a member of a family of Ca(2+) and membrane-binding proteins--to be a marker of ceramide platforms. Using fluorescently tagged annexin A1, we show that, upon its generation within the plasma membrane, ceramide self-associates into platforms that subsequently invaginate and fuse with mitochondria. An accumulation of ceramide within the mitochondria of apoptotic cells was also confirmed using a ceramide-specific antibody. Electron microscopic tomography confirmed that upon the formation of ceramide platforms, the invaginated regions of the plasma membrane extend deep into the cytoplasm forming direct physical contacts with mitochondrial outer membranes. Ceramide might thus be directly transferred from the plasma membrane to the mitochondrial outer membrane. It is conceivable that this "kiss-of-death" increases the permeability of the mitochondrial outer membrane thereby triggering apoptosis.     

Annexins as intracellular calcium sensors

The annexins are a multigene family of Ca(2+)- and charged phospholipid-binding proteins. Although they have been ascribed with diverse functions, there is no consensus about the role played by this family as a whole. We have mapped the Ca(2+)-induced translocations of four members of the annexin family and of two truncated annexins in live cells, and demonstrated that these proteins interact with the plasma membrane as well as with internal membrane systems in a highly coordinated manner. Annexin 2 was the most Ca(2+) sensitive of the studied proteins, followed by annexins 6, 4 and 1. The calcium sensitivity of annexin 2 increased further following co-expression with S100A10. Upon elevation of [Ca(2+)](i), annexins 2 and 6 translocated to the plasma membrane, whereas annexins 4 and 1 also became associated with intracellular membranes and the nuclear envelope. The NH(2)-terminus had a modulatory effect on plasma membrane binding: its truncation increased the Ca(2+) sensitivity of annexin 1, and decreased that of annexin 2. Given the fact that several annexins are present within any one cell, it is likely that they form a sophisticated [Ca(2+)] sensing system, with a regulatory influence on other signaling pathways.     

Stress fibres-a Ca2+ -independent store for annexins?

Annexins belong to a family of lipid-binding proteins that are implicated in membrane organization. Several members are capable of binding to actin and, in smooth muscle cells, annexin 6 is known to form a Ca(2+)-dependent, plasmalemmal complex with actin filaments. Annexins can also associate with F-actin containing stress fibres within cultured smooth muscle cells or fibroblasts in a Ca(2+)-independent manner. Depolymerization of stress-fibre systems with cytochalasin D leads to the translocation of actin-bound annexin 2 from the cytoplasm to the plasma membrane at high intracellular levels of Ca(2+). This type of Ca(2+)-dependent annexin mobility is observed only in cells of mesenchymal phenotype, which have a well-developed stress-fibre system; not in epithelial cells.     

Cholesterol enhances phospholipid binding and aggregation of annexins by their core domain

Annexins are Ca(2+)-dependent phospholipid-binding proteins composed of two domains: A conserved core that is responsible for Ca(2+)- and phospholipid-binding, and a variable N-terminal tail. A Ca(2+)-independent annexin 2-membrane association has been shown to be modulated by the presence of cholesterol in the membranes. Herein, the roles of the core and the N-terminal tail on the cholesterol-enhancement of annexin 2 membrane binding and aggregation were studied. The results show that (i) the cholesterol-mediated increase in membrane binding and in the Ca(2+) sensitivity for membrane aggregation were not modified by a N-terminal peptide (residues 15-26), and were conserved in mutants of the N-terminal end (S11 and S25 substitutions); (ii) cholesterol induced an increase in the Ca(2+)-dependent membrane binding and aggregation of the N-terminally truncated protein (Delta 1-29); and (iii) annexins 5 and 6, two proteins with unrelated N-terminal tails and homologous core domains showed a cholesterol-mediated enhancement of the Ca(2+)-dependent binding to membranes. These data indicate that the core domain is responsible for the cholesterol-mediated effects. A model for the cholesterol effect in membrane organisation, annexin binding and aggregation is discussed.     

Calcium-dependent proapoptotic effect of Taenia solium metacestodes annexin B1 on human eosinophils: a novel strategy to prevent host immune response

Annexins are a family of calcium-dependent phospholipid-binding proteins that have been proposed to be involved in a wide range of important biological processes. At present, only a few annexins have been identified in parasites, and the physiological roles of these annexins are obscure. Earlier, we cloned a novel annexin (annexin B1) from Taenia solium metacestodes and found that annexin B1 was detectable in the surrounding host-derived layer with granulomaous infiltration. The objective of this study was to investigate the secretion and physiological function of annexin B1. We expressed a green fluorescent protein-tagged annexin B1 (GFP-anxB1) in living SiHa cells and showed that it was secreted upon stimulation with dexamethasone (Dex). This secretion was not inhibited by brefeldin A but was blocked by pre-treatment with the intracellular calcium-specific chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Furthermore, we describe for the first time that annexin B1 can bind to the extracellular surface of human eosinophils and produce Ca(2+)-influx. The Ca(2+)-influx induced apoptosis in eosinophils, which was inhibited by pre-loading the Ca(2+) channel blocker 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-metho-xyphenethyl]-1H-imidazole, HCl (SKF-96365). In conclusion, these findings represent direct and substantial evidence for the secretion of annexin B1 by living cells; the apoptosis in eosinophil induced by annexin B1 might be a novel strategy for T. solium metacestodes to prevent the host's immune attack.     

Tailored protection against plasmalemmal injury by annexins with different Ca2+ sensitivities

The annexins, a family of Ca(2+)- and lipid-binding proteins, are involved in a range of intracellular processes. Recent findings have implicated annexin A1 in the resealing of plasmalemmal injuries. Here, we demonstrate that another member of the annexin protein family, annexin A6, is also involved in the repair of plasmalemmal lesions induced by a bacterial pore-forming toxin, streptolysin O. An injury-induced elevation in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) triggers plasmalemmal repair. The highly Ca(2+)-sensitive annexin A6 responds faster than annexin A1 to [Ca(2+)](i) elevation. Correspondingly, a limited plasmalemmal injury can be promptly countered by annexin A6 even without the participation of annexin A1. However, its high Ca(2+) sensitivity makes annexin A6 highly amenable to an unproductive binding to the uninjured plasmalemma; during an extensive injury accompanied by a massive elevation in [Ca(2+)](i), its active pool is severely depleted. In contrast, annexin A1 with a much lower Ca(2+) sensitivity is ineffective at the early stages of injury; however, it remains available for the repair even at high [Ca(2+)](i). Our findings highlight the role of the annexins in the process of plasmalemmal repair; a number of annexins with different Ca(2+)-sensitivities provide a cell with the means to react promptly to a limited injury in its early stages and, at the same time, to withstand a sustained injury accompanied by the continuous formation of plasmalemmal lesions.     

Annexin A4 self-association modulates general membrane protein mobility in living cells

Annexins are Ca2+-regulated phospholipid-binding proteins whose function is only partially understood. Annexin A4 is a member of this family that is believed to be involved in exocytosis and regulation of epithelial Cl- secretion. In this work, fluorescent protein fusions of annexin A4 were used to investigate Ca2+-induced annexin A4 translocation and self-association on membrane surfaces in living cells. We designed a novel, genetically encoded, FRET sensor (CYNEX4) that allowed for easy quantification of translocation and self-association. Mobility of annexin A4 on membrane surfaces was investigated by FRAP. The experiments revealed the immobile nature of annexin A4 aggregates on membrane surfaces, which in turn strongly reduced the mobility of transmembrane and plasma membrane associated proteins. Our work provides mechanistic insight into how annexin A4 may regulate plasma membrane protein function.     

Modes of annexin-membrane interactions analyzed by employing chimeric annexin proteins

Annexin II is a member of the annexin family of Ca(2+)- and phospholipid-binding proteins which is particularly enriched on early endosomal membranes and has been implicated in participating in endocytic events. In contrast to other endosomal annexins the association of annexin II with its target membrane can occur in the absence of Ca(2+) in a manner depending on the unique N-terminal domain of the protein. However, endosome binding of annexin II does not require formation of a protein complex with the intracellular ligand S100A10 (p11) as an annexin II mutant protein (PM AnxII) incapable of interacting with p11 is still present on endosomal membranes. Fusion of the N-terminal sequence of this PM AnxII (residues 1-27) to the conserved protein core of annexin I transfers the capability of Ca(2+)-independent membrane binding to the otherwise Ca(2+)-sensitive annexin I. These results underscore the importance of the N-terminal sequence of annexin II for the Ca(2+)-independent endosome association and argue for a direct interaction of this sequence with an endosomal membrane receptor.     

Atypical properties displayed by annexin A9, a novel member of the annexin family of Ca(2+) and lipid binding proteins

Annexin A9 is a novel member of the annexin family of Ca(2+) and phospholipid binding proteins which has so far only been identified in EST data bases and whose deduced protein sequence shows mutations in residues considered crucial for Ca(2+) coordination in other annexins. To elucidate whether the annexin A9 protein is expressed as such and to characterize its biochemical properties we probed cell extracts with specific anti-annexin A9 antibodies and developed a recombinant expression system. We show that the protein is found in HepG2 hepatoma cell lysates and that a green fluorescent protein-tagged form is abundantly expressed in the cytosol of HeLa cells. Recombinant expression in bacteria yields a soluble protein that can be enriched by conventional chromatographic procedures. The protein is capable of binding phosphatidylserine containing liposomes albeit only at Ca(2+) concentrations exceeding 2 mM. Moreover and in contrast to other annexins this binding appears to be irreversible as the liposome-bound annexin A9 cannot be released by Ca(2+) chelation. These results indicate that annexin A9 is a unique member of the annexin family whose intracellular activity is not subject to Ca(2+) regulation.     

Apical membrane targeting of Nedd4 is mediated by an association of its C2 domain with annexin XIIIb

Nedd4 is a ubiquitin protein ligase (E3) containing a C2 domain, three or four WW domains, and a ubiquitin ligase HECT domain. We have shown previously that the C2 domain of Nedd4 is responsible for its Ca(2+)-dependent targeting to the plasma membrane, particularly the apical region of epithelial MDCK cells. To investigate this apical preference, we searched for Nedd4-C2 domain-interacting proteins that might be involved in targeting Nedd4 to the apical surface. Using immobilized Nedd4-C2 domain to trap interacting proteins from MDCK cell lysate, we isolated, in the presence of Ca(2+), a approximately 35-40-kD protein that we identified as annexin XIII using mass spectrometry. Annexin XIII has two known isoforms, a and b, that are apically localized, although XIIIa is also found in the basolateral compartment. In vitro binding and coprecipitation experiments showed that the Nedd4-C2 domain interacts with both annexin XIIIa and b in the presence of Ca(2+), and the interaction is direct and optimal at 1 microM Ca(2+). Immunofluorescence and immunogold electron microscopy revealed colocalization of Nedd4 and annexin XIIIb in apical carriers and at the apical plasma membrane. Moreover, we show that Nedd4 associates with raft lipid microdomains in a Ca(2+)-dependent manner, as determined by detergent extraction and floatation assays. These results suggest that the apical membrane localization of Nedd4 is mediated by an association of its C2 domain with the apically targeted annexin XIIIb.     

Annexins: linking Ca2+ signalling to membrane dynamics

Eukaryotic cells contain various Ca(2+)-effector proteins that mediate cellular responses to changes in intracellular Ca(2+) levels. A unique class of these proteins - annexins - can bind to certain membrane phospholipids in a Ca(2+)-dependent manner, providing a link between Ca(2+) signalling and membrane functions. By forming networks on the membrane surface, annexins can function as organizers of membrane domains and membrane-recruitment platforms for proteins with which they interact. These and related properties enable annexins to participate in several otherwise unrelated events that range from membrane dynamics to cell differentiation and migration.     

"Nonclassical" secretion of annexin A2 to the lumenal side of the enterocyte brush border membrane

Annexin A2 is a member of the annexin family of Ca(2+)-dependent lipid binding proteins and believed to be engaged in membrane transport processes in a number of cell types. In small intestinal enterocytes, we localized annexin A2 to the brush border region, where it was found mainly on the lumenal side of the microvilli, showing an apical secretion by a "nonclassical" mechanism. In addition, annexin A2 was associated with surface-connected, deep apical tubules in the apical terminal web region and with an underlying pleiomorphic, tubulo-vesicular compartment (subapical compartment/multivesicular bodies). By subcellular fractionation, the 36 kDa full-length form of annexin A2 was approximately equally distributed between the Mg(2+)-precipitated fraction (containing intracellular and basolateral membranes) and the microvillar membrane fraction. In addition, a 33 kDa molecular form of annexin A2 was seen in the latter fraction that could be generated from the full-length annexin A2 by digestion with trypsin. Taken together, the results suggest that annexin A2 acts in exocytic apical membrane trafficking and is proteolytically cleaved in situ by pancreatic proteinases once it has become externalized to the lumenal side of the brush border membrane. On the basis of its well-known membrane fusogenic properties, we propose a model for the nonclassical membrane translocation of annexin A2.     

Annexin 5 mediates a peroxide-induced Ca(2+) influx in B cells

Annexin 5 is a Ca(2+)-binding protein, the function of which is poorly understood. Structural and electrophysiological studies have shown that annexin 5 can mediate Ca(2+) fluxes across phospholipid membranes in vitro [1]. There is, however, no direct evidence for the existence of annexin 5 Ca(2+) channels in living cells. Here, we show that annexin 5 inserts into phospholipid vesicle membranes at neutral pH in the presence of peroxide. We then used targeted gene disruption to explore the role of annexin 5 in peroxide-induced Ca(2+) signaling in DT40 pre-B cells. DT40 clones lacking annexin 5 exhibited normal Ca(2+) responses to both thapsigargin and B-cell receptor stimulation, but lacked the sustained phase of the response to peroxide. This late phase was due to Ca(2+) influx from the extracellular space, demonstrating that annexin 5 mediates a peroxide-induced Ca(2+) influx. Thus, peroxide induces annexin 5 membrane insertion in vitro, and peroxide-induced Ca(2+) entry in vivo in DT40 cells requires annexin 5. Our results are consistent with a role for annexin 5 either as a Ca(2+) channel, or as a signaling intermediate in the peroxide-induced Ca(2+)-influx pathway.     

Ceramide is a potent activator of plasma membrane Ca2+-ATPase from kidney-promixal tubule cells with protein kinase A as an intermediate

The kidney-proximal tubules are involved in reabsorbing two-thirds of the glomerular ultrafiltrate, a key Ca(2+)-modulated process that is essential for maintaining homeostasis in body fluid compartments. The basolateral membranes of these cells have a Ca(2+)-ATPase, which is thought to be responsible for the fine regulation of intracellular Ca(2+) levels. In this paper we show that nanomolar concentrations of ceramide (Cer(50) = 3.5 nm), a natural product derived from sphingomyelinase activity in biological membranes, promotes a 50% increase of Ca(2+)-ATPase activity in purified basolateral membranes. The stimulatory effect of ceramide occurs through specific and direct (cAMP-independent) activation of a protein kinase A (blocked by 10 nm of the specific inhibitor of protein kinase A (PKA), the 5-22 peptide). The activation of PKA by ceramide results in phosphorylation of the Ca(2+)-ATPase, as detected by an anti-Ser/Thr specific PKA substrate antibody. It is observed a straight correlation between increase of Ca(2+)-ATPase activity and PKA-mediated phosphorylation of the Ca(2+) pump molecule. Ceramide also stimulates phosphorylation of renal Ca(2+)-ATPase via protein kinase C, but stimulation of this pathway, which inhibits the Ca(2+) pump in kidney cells, is counteracted by the ceramide-triggered PKA-mediated phosphorylation. The potent effect of ceramide reveals a new physiological activator of the plasma membrane Ca(2+)-ATPase, which integrates the regulatory network of glycerolipids and sphingolipids present in the basolateral membranes of kidney cells.     

Identification of a novel protein complex containing annexin VI, Fyn, Pyk2, and the p120(GAP) C2 domain

p120(GAP) (RasGAP) has been proposed to function as both an inhibitor and effector of Ras. Previously we have shown that RasGAP contains a C2 domain which mediates both Ca(2+)-dependent membrane association and protein-protein interactions. Specifically, three proteins have been isolated in a complex with the C2 domain of RasGAP; these are the Ca(2+)-dependent lipid binding protein annexin VI (p70) and two previously unidentified proteins, p55 and p120. Here we provide evidence that p55 is the Src family kinase Fyn and p120 is the focal adhesion kinase family member Pyk2. In addition, in vitro binding assays indicate that Fyn, but not Pyk2 binds directly to annexin VI. Finally, co-immunoprecipitation studies in Rat-1 fibroblasts confirm that Fyn, Pyk2, annexin VI and RasGAP can form a protein complex in mammalian cells.     

TRPC3 controls agonist-stimulated intracellular Ca2+ release by mediating the interaction between inositol 1,4,5-trisphosphate receptor and RACK1

Activation of TRPC3 channels is concurrent with inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R)-mediated intracellular Ca(2+) release and associated with phosphatidylinositol 4,5-bisphosphate hydrolysis and recruitment to the plasma membrane. Here we report that interaction of TRPC3 with receptor for activated C-kinase-1 (RACK1) not only determines plasma membrane localization of the channel but also the interaction of IP(3)R with RACK1 and IP(3)-dependent intracellular Ca(2+) release. We show that TRPC3 interacts with RACK1 via N-terminal residues Glu-232, Asp-233, Glu-240, and Glu-244. Carbachol (CCh) stimulation of HEK293 cells expressing wild type TRPC3 induced recruitment of a ternary TRPC3-RACK1-IP(3)R complex and increased surface expression of TRPC3 and Ca(2+) entry. Mutation of the putative RACK1 binding sequence in TRPC3 disrupted plasma membrane localization of the channel. CCh-stimulated recruitment of TRPC3-RACK1-IP(3)R complex as well as increased surface expression of TRPC3 and receptor-operated Ca(2+) entry were also attenuated. Importantly, CCh-induced intracellular Ca(2+) release was significantly reduced as was RACK1-IP(3)R association without any change in thapsigargin-stimulated Ca(2+) release and entry. Knockdown of endogenous TRPC3 also decreased RACK1-IP(3)R association and decreased CCh-stimulated Ca(2+) entry. Furthermore, an oscillatory pattern of CCh-stimulated intracellular Ca(2+) release was seen in these cells compared with the more sustained pattern seen in control cells. Similar oscillatory pattern of Ca(2+) release was seen after CCh stimulation of cells expressing the TRPC3 mutant. Together these data demonstrate a novel role for TRPC3 in regulation of IP(3)R function. We suggest TRPC3 controls agonist-stimulated intracellular Ca(2+) release by mediating interaction between IP(3)R and RACK1.     

Sphingomyelinase causes endothelium-dependent vasorelaxation through endothelial nitric oxide production without cytosolic Ca(2+) elevation

Neutral sphingomyelinase (N-SMase) elevated nitric oxide (NO) production without affecting intracellular Ca(2+) concentration ([Ca(2+)](i)) in endothelial cells in situ on aortic valves, and induced prominent endothelium-dependent relaxation of coronary arteries, which was blocked by N(omega)-monomethyl-L-arginine, a NO synthase (NOS) inhibitor. N-SMase induced translocation of endothelial NOS (eNOS) from plasma membrane caveolae to intracellular region, eNOS phosphorylation on serine 1179, and an increase of ceramide level in endothelial cells. Membrane-permeable ceramide (C(8)-ceramide) mimicked the responses to N-SMase. We propose the involvement of N-SMase and ceramide in Ca(2+)-independent eNOS activation and NO production in endothelial cells in situ, linking to endothelium-dependent vasorelaxation.     

Annexin B12 is a sensor of membrane curvature and undergoes major curvature-dependent structural changes

The regulation of membrane curvature plays an important role in many membrane trafficking and fusion events. Recent studies have begun to identify some of the proteins involved in controlling and sensing the curvature of cellular membranes. A mechanistic understanding of these processes is limited, however, as structural information for the membrane-bound forms of these proteins is scarce. Here, we employed a combination of biochemical and biophysical approaches to study the interaction of annexin B12 with membranes of different curvatures. We observed selective and Ca(2+)-independent binding of annexin B12 to negatively charged vesicles that were either highly curved or that contained lipids with negative intrinsic curvature. This novel curvature-dependent membrane interaction induced major structural rearrangements in the protein and resulted in a backbone fold that was different from that of the well characterized Ca(2+)-dependent membrane-bound form of annexin B12. Following curvature-dependent membrane interaction, the protein retained a predominantly alpha-helical structure but EPR spectroscopy studies of nitroxide side chains placed at selected sites on annexin B12 showed that the protein underwent inside-out refolding that brought previously buried hydrophobic residues into contact with the membrane. These structural changes were reminiscent of those previously observed following Ca(2+)-independent interaction of annexins with membranes at mildly acidic pH, yet they occurred at neutral pH in the presence of curved membranes. The present data demonstrate that annexin B12 is a sensor of membrane curvature and that membrane curvature can trigger large scale conformational changes. We speculate that membrane curvature could be a physiological signal that induces the previously reported Ca(2+)-independent membrane interaction of annexins in vivo.     

STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx

Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.     

Involvement of ceramide in hyperosmotic shock-induced death of erythrocytes

Erythrocytes lack nuclei and mitochondria, the organelles important for apoptosis of nucleated cells. However, following increase of cytosolic Ca(2+) activity, erythrocytes undergo cell shrinkage, cell membrane blebbing and breakdown of phosphatidylserine asymmetry, all features typical for apoptosis in nucleated cells. The same events are observed following osmotic shock, an effect mediated in part by activation of Ca(2+)-permeable cation channels. However, erythrocyte death following osmotic shock is blunted but not prevented in the absence of extracellular Ca(2+) pointing to additional mechanisms. As shown in this study, osmotic shock (950 mOsm) triggers sphingomyelin breakdown and formation of ceramide. The stimulation of annexin binding following osmotic shock is mimicked by addition of ceramide or purified sphingomyelinase and significantly blunted by genetic (aSM-deficient mice) or pharmacologic (50 microM 3,4-dichloroisocoumarin) knockout of sphingomyelinase. The effect of ceramide is blunted but not abolished in the absence of Ca(2+). Conversely, osmotic shock-induced annexin binding is potentiated in the presence of sublethal concentrations of ceramide. In conclusion, ceramide and Ca(2+) entry through cation channels concert to trigger erythrocyte death during osmotic shock.