Advertisement

Erythropoiesis pp 205-228 | Cite as

High-Resolution Fluorescence Microscope Imaging of Erythroblast Structure

  • Alyson S. Smith
  • Roberta B. Nowak
  • Velia M. Fowler
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1698)

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.

Key words

Erythroblast Fetal liver Bone marrow Spleen Immunofluorescence Confocal microscopy 

References

  1. 1.
    Palis J (2014) Primitive and definitive erythropoiesis in mammals. Front Physiol 5:3CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ji P, Murata-Hori M, Lodish H (2011) Formation of mammalian erythrocytes: chromatin condensation and enucleation. Trends Cell Biol 21:409–415CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Migliaccio AR (2010) Erythroblast enucleation. Haematologica 95:1985–1988CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Liu J, Guo X, Mohandas N et al (2010) Membrane remodeling during reticulocyte maturation. Blood 115:2021–2027CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    An X, Schulz VP, Li J et al (2014) Global transcriptome analyses of human and murine terminal erythroid differentiation. Blood 123:3466–3477CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Bell AJ, Satchwell TJ, Heesom KJ et al (2013) Protein distribution during human erythroblast enucleation in vitro. PLoS One 8:e60300CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    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–e138CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Hattangadi SM, Wong P, Zhang L, Flygare J, Lodish HF (2011) From stem cell to red cell: regulation of erythropoiesis at multiple levels. Blood 118:6258–6268CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Paulson RF, Shi L, Wu DC (2011) Stress erythropoiesis: new signals and new stress progenitor cells. Curr Opin Hematol 18:139–145CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Chasis JA, Mohandas N (2008) Erythroblastic islands: niches for erythropoiesis. Blood 112:470–478CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Manawani D, Bieker JJ (2008) The erythroblastic island, Chapter 2. Curr Top Dev Biol 82:23CrossRefGoogle Scholar
  12. 12.
    Lee JC-M, Gimm JA, Lo AJ et al (2004) Mechanism of protein sorting during erythroblast enucleation: role of cytoskeletal connectivity. Blood 103:1912–1919CrossRefPubMedGoogle Scholar
  13. 13.
    Konstantinidis DG, Pushkaran S, Johnson JF et al (2012) Signaling and cytoskeletal requirements in erythroblast enucleation. Blood 119:6118–6127CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Koury ST, Koury MJ, Bondurant MC (1989) Cytoskeletal distribution and function during the maturation and enucleation of mammalian erythroblasts. J Cell Biol 109:3005–3013CrossRefPubMedGoogle Scholar
  15. 15.
    Pawley JB (ed) (2006) Handbook of biological confocal microscopy. Springer, New York, NYGoogle Scholar
  16. 16.
    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):1144–1155CrossRefPubMedGoogle Scholar
  17. 17.
    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 123:758–767CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Gokhin DS, Fowler VM (2011) Cytoplasmic γ-actin and tropomodulin isoforms link to the sarcoplasmic reticulum in skeletal muscle fibers. J Cell Biol 194:105–120CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Fischer AH, Jacobson KA, Rose J, Zeller R (2008) Preparation of slides and coverslips for microscopy. Cold Spring Harb Protoc 2008:pdb.prot4988–pdb.prot4988Google Scholar
  20. 20.
    Waterman-Storer CM (2001) Microtubule/organelle motility assays. Curr Protoc Cell Biol. Chapter 13, Unit 13.1Google Scholar
  21. 21.
    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 86:3993–4003CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    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 23:283–297PubMedGoogle Scholar
  23. 23.
    Latunde-Dada GO, McKie AT, Simpson RJ (2006) Animal models with enhanced erythropoiesis and iron absorption. Biochim Biophys Acta 1762:414–423CrossRefPubMedGoogle Scholar
  24. 24.
    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 215:281–296CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Alyson S. Smith
    • 1
  • Roberta B. Nowak
    • 1
  • Velia M. Fowler
    • 1
  1. 1.Department of Molecular MedicineThe Scripps Research InstituteLa JollaUSA

Personalised recommendations