Preparation and characterization of an endogenously fluorescent annexin for detection of apoptotic cells.
Annexin proteins specifically bind anionic phospholipids such as phosphatidylserine, which are normally confined to the cytoplasmic leaflet of cellular membranes. During programmed cell death, or apoptosis, this phospholipid asymmetry is lost, and anionic phospholipids are exposed on the extracellular leaflet of the plasma membrane where they are accessible to exogenously added, labeled annexins. Chemically [e.g., fluoroscein isothiocyanate (FITC)]-modified annexin V has been widely used to detect and enumerate apoptotic cells by flow cytometry. We prepared chimeric proteins containing green fluorescent protein (GFP) fused to annexin V. A chimera containing GFP fused to the C-terminus of annexin V was soluble and fluorescent, but was unable to bind phospholipids. In contrast, a chimera containing GFP fused to the N-terminus of annexin V specifically bound apoptotic cells. GFP-annexin V represents a sensitive and facile alternative to FITC-annexin V for studies of apoptosis.
Ernst
J D
JD
Division of Infectious Diseases, University of California, San Francisco 94143, USA. joel@cgl.ucsf.edu
Yang
L
L
Rosales
J L
JL
Broaddus
V C
VC
D000818:Animals; D017304:Annexin A5 / Q000235:genetics*; D017209:Apoptosis; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics*; D008563:Membrane Lipids / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010994:Pleura / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013050:Spectrometry, Fluorescence
D017304:Annexin A5; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008563:Membrane Lipids; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins