Abstract
During erythropoiesis, erythroblasts undergo dramatic morphological changes to produce mature erythrocytes. Many unanswered questions regarding the molecular mechanisms behind these changes can be addressed with high-resolution fluorescence imaging. Immunofluoresence staining enables localization of specific molecules, organelles, and membrane components in intact cells at different phases of erythropoiesis. Confocal laser scanning microscopy can provide high-resolution, three-dimensional images of stained structures, which can be used to dissect the molecular mechanisms driving erythropoiesis. The sample preparation, staining procedure, imaging parameters, and image analysis methods used directly affect the quality of the confocal images and the amount and accuracy of information that they can provide. Here, we describe methods to dissect erythropoietic tissues from mice, to perform immunofluorescence staining and confocal imaging of various molecules, organelles and structures of interest in erythroblasts, and to present and quantitatively analyze the data obtained in these fluorescence images.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Palis J (2014) Primitive and definitive erythropoiesis in mammals. Front Physiol 5:3
Ji P, Murata-Hori M, Lodish H (2011) Formation of mammalian erythrocytes: chromatin condensation and enucleation. Trends Cell Biol 21:409–415
Migliaccio AR (2010) Erythroblast enucleation. Haematologica 95:1985–1988
Liu J, Guo X, Mohandas N et al (2010) Membrane remodeling during reticulocyte maturation. Blood 115:2021–2027
An X, Schulz VP, Li J et al (2014) Global transcriptome analyses of human and murine terminal erythroid differentiation. Blood 123:3466–3477
Bell AJ, Satchwell TJ, Heesom KJ et al (2013) Protein distribution during human erythroblast enucleation in vitro. PLoS One 8:e60300
Wong P, Hattangadi SM, Cheng AW et al (2011) Gene induction and repression during terminal erythropoiesis are mediated by distinct epigenetic changes. Blood 118:e128–e138
Hattangadi SM, Wong P, Zhang L, Flygare J, Lodish HF (2011) From stem cell to red cell: regulation of erythropoiesis at multiple levels. Blood 118:6258–6268
Paulson RF, Shi L, Wu DC (2011) Stress erythropoiesis: new signals and new stress progenitor cells. Curr Opin Hematol 18:139–145
Chasis JA, Mohandas N (2008) Erythroblastic islands: niches for erythropoiesis. Blood 112:470–478
Manawani D, Bieker JJ (2008) The erythroblastic island, Chapter 2. Curr Top Dev Biol 82:23
Lee JC-M, Gimm JA, Lo AJ et al (2004) Mechanism of protein sorting during erythroblast enucleation: role of cytoskeletal connectivity. Blood 103:1912–1919
Konstantinidis DG, Pushkaran S, Johnson JF et al (2012) Signaling and cytoskeletal requirements in erythroblast enucleation. Blood 119:6118–6127
Koury ST, Koury MJ, Bondurant MC (1989) Cytoskeletal distribution and function during the maturation and enucleation of mammalian erythroblasts. J Cell Biol 109:3005–3013
Pawley JB (ed) (2006) Handbook of biological confocal microscopy. Springer, New York, NY
Nowak RB, Papoin J, Gokhin DS, Casu C, Rivella S, Lipton JM, Blanc L, Fowler VM (2017) Tropomodulin 1 controls erythroblast enucleation via regulation of F-actin in the enucleosome. Blood 130(9):1144–1155
Sui Z, Nowak RB, Bacconi A, Kim NE, Liu H, Li J, Wickrema A, An X, Fowler VM (2014) Tropomodulin3-null mice are embryonic lethal with anemia due to impaired erythroid terminal differentiation in the fetal liver. Blood 123:758–767
Gokhin DS, Fowler VM (2011) Cytoplasmic γ-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers. J Cell Biol 194:105–120
Fischer AH, Jacobson KA, Rose J, Zeller R (2008) Preparation of slides and coverslips for microscopy. Cold Spring Harb Protoc 2008:pdb.prot4988–pdb.prot4988
Waterman-Storer CM (2001) Microtubule/organelle motility assays. Curr Protoc Cell Biol. Chapter 13, Unit 13.1
Costes SV, Daelemans D, Cho EH, Dobbin Z, Pavlakis G, Lockett S (2004) Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys J 86:3993–4003
Pantel K, Loeffler M, Bungart B, Wichmann HE (1990) A mathematical model of erythropoiesis in mice and rats. Part 4: Differences between bone marrow and spleen. Cell Tissue Kinet 23:283–297
Latunde-Dada GO, McKie AT, Simpson RJ (2006) Animal models with enhanced erythropoiesis and iron absorption. Biochim Biophys Acta 1762:414–423
Bernas T, ZarÉBski M, Cook RR, Dobrucki JW (2004) Minimizing photobleaching during confocal microscopy of fluorescent probes bound to chromatin: role of anoxia and photon flux. J Microsc 215:281–296
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Smith, A.S., Nowak, R.B., Fowler, V.M. (2018). High-Resolution Fluorescence Microscope Imaging of Erythroblast Structure. In: Lloyd, J. (eds) Erythropoiesis. Methods in Molecular Biology, vol 1698. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7428-3_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7428-3_12
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7427-6
Online ISBN: 978-1-4939-7428-3
eBook Packages: Springer Protocols