Annexins

Annexins form an evolutionary conserved family of Ca2+ and phospholipid binding proteins implicated in membrane trafficking and the regulation of Ca2+ currents across membranes. 

Non-EF-hand Ca2+-binding proteins including the annexins, C2-domain proteins, and gelsolin, and the EF-hand Ca2+-binding proteins. 

ALG-2 is the fust calcium-binding protein of the EF-hand family found to be directly involved in apoptosis. ALG-2 is a 22 kDa protein and like the other members of the penta EF-hand family, contains five EF-hands, with only two of them functional. ALG-2 protein is expressed in the brain and eye and was found to be upregulated in various cancer tissues. Several targets have been found, such as proteins AEP, Alix, preflin, and annexins, suggesting a putative role of ALG-2 in apoptosis. 

Gavins FN, Leoni G, Getting SJ (2006) Annexin 1 and melanocoitin peptide therapy for protection against ischaemic-reperfusion damage in the heart. Scientific-WorldJournal 6 1008-1023... 

Annexin V PT UOS staining is another procedure which allows to label the cells by allowing the observation of the morphological features of apoptosis. 

One of the early events of the apoptotic process involves the translocation of phosphatidylserine on the surface of cell membranes annexin V binding and propidium iodide uptake reveals various cellular states. After treatment with organotin(IV) compounds the cells could be categorized into populations vital cells (annexin V /P ), early apoptotic cells (annexin V /P ), late apoptotic cells (annexin V /P ), and necrotic cells (annexin V /P" ). Cells are observed with a fluorescence microscope and it is possible to observe translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer one and to see a green stain for annexin V FLUOS bound to PS, and a red stain for propidium iodide. 

CLARKE R G, LUND E K, JOHNSON I T aud PINDER A c (2000) Apoptosis cau he detected in attached colonic adenocarcinoma HT29 cells using annexin V binding, hut uot hy TUNEL assay or suh-GO DNA content . Cytometry, 39 141-50. 

Fig. 13. The binding sites of calcium in (a) parvalbumin (41a), (b) annexin (41) and (c) calmodulin (42). The drawings show two bidentate carboxylates coordinated to Ca2 in the EF-hand site of parvalbumin, and one bidentate carboxylate coordinated to Ca2 in annexin and calmodulin. All the donor atoms coordinated to the calciums are oxygen donor atoms from carboxylates of asp = aspartate, or glu = glutamate, or else peptide carbonyl oxygens from gly = glycine or met = methionine. Redrawn after Refs. (41-42).

Uittenbogaard, A, Everson, WV, Matveev, SV, and Smart, EJ, 2002. Cholesteryl ester is transported from caveolae to internal membranes as part of a caveolin-annexin II lipid-protein complex. J Biol Chem 277,4925—4-931. 

Hwang and Bowen (2005b) Tomato paste hexane extract Apoptosis by Annexin V binding... 

Necrosis by LDH Mitochondrial transmembrane potential by DiOC6 and JC-1 fluorescence Apoptosis by cytochrome c release Annexin V binding DNA fragmentation by agarose gel electrophoresis... 

Annexins Internal associated with lipids, trigger... 

The upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system is described. By off-axis coupling of the intensity-modulated laser from a fiber and using a high numerical aperture oil objective, TIR-FLIM can be readily achieved. The usefulness of the technique is demonstrated by a fluorescence resonance energy transfer study of Annexin A4 relocation and two-dimensional crystal formation near the plasma membrane of cultured mammalian cells. Possible future applications and comparison to other techniques are discussed. 

To demonstrate an application of TIRF-FLIM, a FRET study of annexin A4 translocation and self-aggregation near the plasma membrane is shown in Fig. 9.4. This is a particularly useful application of TIRF-FLIM, since TIRF provides the spatial contrast of detecting only molecules immediately adjacent to the plasma membrane and the lifetime contrast reports on the aggregation state of annexin A4. Annexins are calcium-dependent lipid-binding domains with a different type of lipid binding domain compared to the common C2 domains (e.g., found in protein kinase C). Annex-ins consist of an N-terminal domain and a core domain binding calcium and phospholipids. The core domain is conserved in the... 

In Fig. 9.4, the results are shown of an experiment using a double (annexinA4-EYFP+ annexinA4-mCherry) transfected cell immediately after addition of the calcium ionophore ionomycin (top) and 5 min after application of ionomycin (bottom). Clearly visible is the diffuse localization of annexin A4 before relocation and the more structured localization (into membrane ruffles and filopodia) after relocation. More importantly, the EYFP fluorescence lifetime is quenched from 2.9 ns before translocation to... 

Piljic, A. and Schultz, C. (2006). Annexin A4 self-association modulates general membrane protein mobility in living cells. Mol. Biol. Cell 17, 3318-28. 

Annexin V-functionalized crosslinked iron oxide (CLIO) was designed as a contrast agent for MRI, which was additionally labeled with Cy5.5 to allow colocalization with optical imaging techniques [98]. Alternatively, conjugation of multiple Gd-DTPA molecules or SPIO particles to the C2 domain of synaptotagmin I was shown to allow the detection of apoptotic cells in vitro [99]. Zhao et al. [100] were the first to apply a C2 domain-functionalized SPIO and showed very promising results for future in vivo applications of MR contrast agents for the detection of apoptotic sites. 

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