In vitro measurement of cell death with the annexin A5 affinity assay

Genderen, Hugo van; Kenis, Heidi; Lux, Petra; Ungeth, Lisette; Maassen, Cecile; Deckers, Niko; Narula, Jagat; Hofstra, Leo; Reutelingsperger, Chris

doi:10.1038/nprot.2006.55

Cited by 86 publications

(67 citation statements)

References 18 publications

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“…Multiple proteins have been implicated as binding partners for cell surface-presented PS and among those annexin A5 is widely accepted as a specific ligand and used as a marker for cell death (11)(12)(13). Surprisingly, prior to this study relatively little was known about the role of other members of the annexin family in cell death recognition.…”

Section: Discussionmentioning

confidence: 99%

“…Several PS-binding serum proteins like protein S (8), milk fat globule protein (MFG-E8) (9) and ␤2-glycoprotein-1 (10) can act as bridging molecules that bind to the signature of the apoptotic cells. The interpretation of the PS signature in the context of bridging molecules and receptors by phagocytes then modulates the response toward apoptotic cells.Annexin A5 (Anxa5-gene, AnxA5-protein) was identified as a specific interaction partner for PS and is extensively used to detect cell death in vitro and in vivo (11)(12)(13). This protein…”

mentioning

confidence: 99%

“…Annexin A5 (Anxa5-gene, AnxA5-protein) was identified as a specific interaction partner for PS and is extensively used to detect cell death in vitro and in vivo (11)(12)(13). This protein…”

mentioning

confidence: 99%

“…Annexin A5 (Anxa5-gene, AnxA5-protein) was identified as a specific interaction partner for PS and is extensively used to detect cell death in vitro and in vivo (11)(12)(13). This protein can also inhibit the phagocytosis of dying cells (14,15) and modulates the cytokine response of phagocytes toward apoptotic and necrotic cells (16).…”

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confidence: 99%

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Identification of Novel Binding Partners (Annexins) for the Cell Death Signal Phosphatidylserine and Definition of Their Recognition Motif

Rosenbaum

Kreft

Etich

et al. 2011

Journal of Biological Chemistry

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Identification and clearance of apoptotic cells prevents the release of harmful cell contents thereby suppressing inflammation and autoimmune reactions. Highly conserved annexins may modulate the phagocytic cell removal by acting as bridging molecules to phosphatidylserine, a characteristic phagocytosis signal of dying cells. In this study five members of the structurally and functionally related annexin family were characterized for their capacity to interact with phosphatidylserine and dying cells. The results showed that AnxA3, AnxA4, AnxA13, and the already described interaction partner AnxA5 can bind to phosphatidylserine and apoptotic cells, whereas AnxA8 lacks this ability. Sequence alignment experiments located the essential amino residues for the recognition of surface exposed phosphatidylserine within the calcium binding motifs common to all annexins. These amino acid residues were missing in the evolutionary young AnxA8 and when they were reintroduced by site directed mutagenesis AnxA8 gains the capability to interact with phosphatidylserine containing liposomes and apoptotic cells. By defining the evolutionary conserved amino acid residues mediating phosphatidylserine binding of annexins we show that the recognition of dying cells represent a common feature of most annexins. Hence, the individual annexin repertoire bound to the cell surface of dying cells may fulfil opsonin-like function in cell death recognition.Discrimination of viable from dying cells is a prerequisite for the efficient clearance of dying and dead cells and to suppress uncontrolled cell lysis and the release of potentially harmful cellular compounds to the local environment. During development and under normal physiological conditions, cells are removed through the process of apoptosis and undergo a strictly defined series of morphological and biochemical changes, before being engulfed by professional phagocytes such as macrophages or even by neighboring cells. A signature of specific "eat me" signals are exposed at the cell surface of apoptotic cells, which enable direct or indirect interactions with phagocytes and dying cells to promote the efficient clearance of apoptotic cells. This reduces the risk of accumulation of secondarily necrotic cells and the release of cellular content to the microenvironment. Exposure of apoptotic-cell associated molecular patterns together with recognition and interpretation of these by phagocytes are crucial steps in the appropriate response of phagocytes toward the engulfed cells (1).A hallmark of early apoptotic cells is the loss of membrane asymmetry and the exposure of anionic phosphatidylserine (PS) 3 on the outer lipid layer of the cells. In most cells PS predominantly resides in the inner leaflet of the plasma membrane, regulating membrane charge and protein localization (2). This membrane asymmetry is actively maintained by an inward transporting aminophospholipid translocase (3). Upon induction of apotosis, rising cytoplasmic Ca 2ϩ levels cause a loss of translocase activity and an activ...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

“…Annexin A5 (Anxa5-gene, AnxA5-protein) was identified as a specific interaction partner for PS and is extensively used to detect cell death in vitro and in vivo (11)(12)(13). This protein…”

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Identification of Novel Binding Partners (Annexins) for the Cell Death Signal Phosphatidylserine and Definition of Their Recognition Motif

Rosenbaum

Kreft

Etich

et al. 2011

Journal of Biological Chemistry

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“…16 Although special assay conditions have been developed to detect PS on the surface of these apoptotic cells, 8 the biological basis for the diminished PS externalization is unclear.…”

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confidence: 99%

Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm

et al. 2012

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Phosphatidylserine (PS) exposure on the external leaflet of the plasma membrane is widely observed during apoptosis and forms the basis for the annexin V binding assay to detect apoptotic cell death. Current efforts to explain PS exposure focus on two potential mechanisms, activation of a phospholipid scramblase or calcium-mediated trafficking of lysosomes to the cell surface. Here, we provide evidence that apoptotic PS exposure instead reflects bidirectional trafficking of membrane between the cell surface and cytoplasm. Using a series of cell lines, some of which expose large amounts of PS during apoptosis and some of which do not, we demonstrate that accumulation of plasma membrane-derived cytoplasmic vesicles in a dynamin-, clathrin-and Cdc42-independent manner is a previously undescribed but widely occurring feature of apoptosis. The apoptotic exposure of PS occurs when these vesicles traffic back to cell surface in a calcium-dependent process that is deficient in a substantial fraction of human cancer cell lines. These observations provide a new model for PS externalization during apoptosis and simultaneously identify an altered step that accounts for the paucity of apoptotic PS exposure in many cell lines. Cell Death and Differentiation (2013) 20, 64-76; doi:10.1038/cdd.2012; published online 3 August 2012A common feature of apoptosis from Caenorhabditis elegans to man is the transfer of phosphatidylserine (PS) and phosphatidylethanolamine, which ordinarily reside on the cytoplasmic surface of the membranes, to the cell surface.

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Nuclear cytochrome c – a mitochondrial visitor regulating damaged chromatin dynamics

et al. 2018

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Over the past decade, evidence has emerged suggesting a broader role for cytochrome c (Cyt c) in programmed cell death. Recently, we demonstrated the ability of Cyt c to inhibit the nucleosome assembly activity of histone chaperones SET/template-activating factor Iβ and NAP1-related protein during DNA damage in humans and plants respectively. Here, we hypothesise a dual concentration-dependent function for nuclear Cyt c in response to DNA damage. We propose that low levels of highly cytotoxic DNA lesions - such as double-strand breaks - induce nuclear translocation of Cyt c, leading to the attenuation of nucleosome assembly and, thereby, increasing the time available for DNA repair. If DNA damage persists or is exacerbated, the nuclear Cyt c concentration would exceed a given threshold, causing the haem protein to block DNA remodelling altogether.

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In vitro measurement of cell death with the annexin A5 affinity assay

Cited by 86 publications

References 18 publications

Identification of Novel Binding Partners (Annexins) for the Cell Death Signal Phosphatidylserine and Definition of Their Recognition Motif

Identification of Novel Binding Partners (Annexins) for the Cell Death Signal Phosphatidylserine and Definition of Their Recognition Motif

Phosphatidylserine exposure during apoptosis reflects bidirectional trafficking between plasma membrane and cytoplasm

Nuclear cytochrome c – a mitochondrial visitor regulating damaged chromatin dynamics

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