Abstract
Normal and dying cells release various types of membrane-bound vesicles including microvesicles, exosomes, and apoptotic bodies. These vesicles play important roles in intercellular communication and signal transduction. However, their diverse forms and subtypes fluctuate in size and other properties. In result current purification approaches do not fully discriminate between different categories of extracellular vesicles. Here, we present a fluorescence technique that specifically identifies apoptotic bodies in preparations of microvesicles, exosomes, and other extracellular vesicles.
The approach exclusively labels the vesicles that contain DNA with 5′PO4 blunt-ended DNA breaks, such as those produced by the apoptotic CAD nuclease during apoptotic DNA degradation. The technique can be useful in studies of apoptosis involving microvesicles and exosomes.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Zaborowski MP, Balaj L, Breakefield XO et al (2015) Extracellular vesicles: composition, biological relevance, and methods of study. Bioscience 65(8):783–797
Poon IK, Lucas CD, Rossi AG et al (2014) Apoptotic cell clearance: basic biology and therapeutic potential. Nat Rev Immunol 14(3):166–180. doi:10.1038/nri3607
Kerr JFR, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239–257
Depraetere V (2000) “Eat me” signals of apoptotic bodies. Nat Cell Biol 2(6):E104
Ravichandran KS (2010) Find-me and eat-me signals in apoptotic cell clearance: progress and conundrums. J Exp Med 207:1807–1817
Akers JC, Gonda D, Kim R et al (2013) Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies. J Neurooncol 113(1):1–11. doi:10.1007/s11060-013-1084-8
Ihara T, Yamamoto T, Sugamata M et al (1998) The process of ultrastructural changes from nuclei to apoptotic body. Virchows Arch 433(5):443–447
Hristov M, Erl W, Linder S et al (2004) Apoptotic bodies from endothelial cells enhance the number and initiate the differentiation of human endothelial progenitor cells in vitro. Blood 104(9):2761–2766
Mathivanan S, Ji H, Simpson RJ (2010) Exosomes: extracellular organelles important in intercellular communication. J Proteomics 73(10):1907–1920. doi:10.1016/j.jprot.2010.06.006
Simpson RJ, Mathivanan S (2012) Extracellular microvesicles: the need for internationally recognised nomenclature and stringent purification criteria. J Proteomics Bioinform 5:ii. doi:10.4172/jpb.10000e10
Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 200(4):373–383
Thery C, Boussac M, Veron P et al (2001) Proteomic analysis of dendritic cell–derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol 166:7309–7318
Crescitelli R, Lässer C, Szabo TG et al (2013) Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes. J Extracell Vesicles 2 (art. 20677). doi:10.3402/jev.v2i0.20677
Didenko VV (2002) Detection of specific double-strand DNA breaks and apoptosis in situ using T4 DNA ligase. Methods Mol Biol 203:143–151
Didenko VV, Minchew CL, Shuman S et al (2004) Semi-artificial fluorescent molecular machine for DNA damage detection. Nano Lett 4(12):2461–2466
Staley K, Blaschke A, Chun J (1997) Apoptotic DNA fragmentation is detected by a semiquantitative ligation-mediated PCR of blunt DNA ends. Cell Death Differ 4:66–75
Hornsby PJ, Didenko VV (2011) In situ ligation: a decade and a half of experience. Methods Mol Biol 682:49–63. doi:10.1007/978-1-60327-409-8_5
Widlak P, Li P, Wang X et al (2000) Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on naked DNA and chromatin substrates. J Biol Chem 275(11):8226–8232
Minchew CL, Didenko VV (2014) Nanoblinker: Brownian motion powered bio-nanomachine for FRET detection of phagocytic phase of apoptosis. PLoS One 9(9):e108734. doi:10.1371/journal.pone.0108734
Minchew CL, Didenko VV (2011) Fluorescent probes detecting the phagocytic phase of apoptosis: enzyme-substrate complexes of topoisomerase and DNA. Molecules 16(6):4599–4614. doi:10.3390/molecules16064599
Didenko VV (2011) 5′OH DNA breaks in apoptosis and their labeling by topoisomerase-based approach. Methods Mol Biol 682:77–87. doi:10.1007/978-1-60327-409-8_7
Samejima K, Earnshaw WC (2005) Trashing the genome: the role of nucleases during apoptosis. Nat Rev Mol Cell Biol 6:677–688
Minchew CL, Didenko VV (2012) In vitro assembly of semi-artificial molecular machine and its use for detection of DNA damage. J Vis Exp 59:e3628. doi:10.3791/3628
Didenko VV, Ngo H, Minchew CL et al (2002) Caspase-3-dependent and -independent apoptosis in focal brain ischemia. Mol Med 8(7):347–352
Didenko VV, Ngo H, Baskin DS (2003) Early necrotic DNA degradation: presence of blunt-ended DNA breaks, 3′ and 5′ overhangs in apoptosis, but only 5′ overhangs in early necrosis. Am J Pathol 162(5):1571–1578
Acknowledgment
This research was supported by grant R01 NS082553 from the National Institute of Neurological Disorders and Stroke, National Institutes of Health and by grants R21 CA178965 from the National Cancer Institute, National Institutes of Health and R21 AR066931 National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health (all to V.V.D.).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Hauser, P., Wang, S., Didenko, V.V. (2017). Apoptotic Bodies: Selective Detection in Extracellular Vesicles. In: Kalyuzhny, A. (eds) Signal Transduction Immunohistochemistry. Methods in Molecular Biology, vol 1554. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6759-9_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6759-9_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6757-5
Online ISBN: 978-1-4939-6759-9
eBook Packages: Springer Protocols