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Electron Tomography and Correlative Approaches in Platelet Studies

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Platelets and Megakaryocytes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1812))

Abstract

Blood platelets play a central role in the arrest of bleeding and the development of thrombosis. Unraveling the complex processes of platelet biogenesis from megakaryocytes, platelet adhesion, aggregation, and secretory responses are important topics in the field of hemostasis and thrombosis. Analysis of the ultrastructural changes that occur during these processes is essential for understanding the rapid membrane dynamics and has contributed substantially to our present knowledge of platelet formation and functioning. Recent developments in real-time imaging, correlative light and electron microscopy imaging (CLEM), and 3D (cryo) electron microscopy and tomography offer exciting opportunities to improve studies of the platelet adhesive responses and secretion at the ultrastructural level in a close to native environment. In this chapter we discuss and illustrate cryo preparation techniques (high-pressure freezing, vitrification), correlative LM and EM workflows, and 3D cryo-electron tomography that we apply in our current research projects.

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Abbreviations

EM:

Electron microscopy

ET:

Electron tomography

CLEM:

Correlative light and electron microscopy

FS:

Freeze substitution

HPF:

High-pressure freezing

LN2:

Liquid nitrogen

CCD:

Charge coupled device

3D:

Three-dimensional

vWF:

von Willebrand factor

SIRT:

Simultaneous iterative reconstruction technique

Missing Wedge:

Missing information due to limited tilt angles

Contouring:

Manual drawing of contour lines in slices of a tomogram

Tomogram:

Computed 3D volume reconstruction of a specimen by using multiple projection images

References

  1. Heijnen HFG, Korporaal SJA (2017) Platelet morphology and ultrastructure. In: Gresele P et al (eds) Platelets in thrombotic and non-thrombotic disorders, pathophysiology, pharmacology and therapeutics. Springer International Publishing AG, Basel, pp 21–37

    Chapter  Google Scholar 

  2. Behnke O (1968) An electron microscope study of the megacaryocyte of the rat bone marrow. I. The development of the demarcation membrane system and the platelet surface coat. J Ultrastruct Res 24(5):412–433

    Article  CAS  PubMed  Google Scholar 

  3. Behnke O (1968) Electron microscopical observations on the surface coating of human blood platelets. J Ultrastruct Res 24(1–2):51–69

    Article  CAS  PubMed  Google Scholar 

  4. Behnke O (1970) The morphology of blood platelet membrane systems. Ser Haematol 3(4):3–16

    PubMed  CAS  Google Scholar 

  5. Behnke O, Forer A (1998) From megakaryocytes to platelets: platelet morphogenesis takes place in the bloodstream. Eur J Haematol Suppl 61:3–23

    PubMed  CAS  Google Scholar 

  6. Morgenstern E (1982) Coated membranes in blood platelets. Eur J Cell Biol 26(2):315

    PubMed  CAS  Google Scholar 

  7. Bentfeld-Barker ME, Bainton DF (1982) Identification of primary lysosomes in human megakaryocytes and platelets. Blood 59(3):472–481

    PubMed  CAS  Google Scholar 

  8. Stenberg PE, Shuman MA, Levine SP, Bainton DF (1984) Redistribution of alpha-granules and their contents in thrombin-stimulated platelets. J Cell Biol 98(2):748–760

    Article  CAS  PubMed  Google Scholar 

  9. White JG (1968) The substructure of human platelet microtubules. Blood 32(4):638–648

    PubMed  CAS  Google Scholar 

  10. White JG (1968) Fine structural alterations induced in platelets by adenosine diphosphate. Blood 31(5):604–622

    PubMed  CAS  Google Scholar 

  11. White JG, Clawson CC (1980) The surface-connected canalicular system of blood platelets–a fenestrated membrane system. Am J Pathol 101(2):353–364

    PubMed  PubMed Central  CAS  Google Scholar 

  12. White JG (1998) Use of the electron microscope for diagnosis of platelet disorders. Semin Thromb Hemost 24(2):163–168

    Article  CAS  PubMed  Google Scholar 

  13. Morgenstern E (1991) Aldehyde fixation causes membrane vesiculation during platelet exocytosis: a freeze-substitution study. Scanning Microsc Suppl 5(4):S109–S115

    PubMed  CAS  Google Scholar 

  14. Murk JLAN, Posthuma G, Koster AJ, Geuze HJ, Verkleij AJ, Kleijmeer MJ, Humbel BM (2003) Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography. J Microsc 212(1):81–90

    Article  CAS  PubMed  Google Scholar 

  15. Posthuma G, Slot JW, Geuze HJ (1987) Usefulness of the immunogold technique in quantitation of a soluble protein in ultra-thin sections. J Histochem Cytochem 35(4):405–410

    Article  CAS  PubMed  Google Scholar 

  16. Tokuyasu KT, Singer SJ (1976) Improved procedures for immunoferritin labeling of ultrathin frozen sections. J Cell Biol 71(3):894–906

    Article  CAS  PubMed  Google Scholar 

  17. Liou W, Geuze HJ, Slot JW (1996) Improving structural integrity of cryosections for immunogold labeling. Histochem Cell Biol 106(1):41–58

    Article  CAS  PubMed  Google Scholar 

  18. Slot JW, Geuze HJ (2007) Cryosectioning and immunolabeling. Nat Protoc 2(10):2480

    Article  CAS  PubMed  Google Scholar 

  19. Riehle U, Hochli M (1973) In: Benedetti EL, Favard P (eds) In freeze-etching technique and applications. Society Francaise de Microscopy Electronique, Paris, pp 31–66

    Google Scholar 

  20. Studer D, Graber W, Al-Amoudi A, Eggli P (2001) A new approach for cryofixation by high-pressure freezing. J Microsc 203(3):285–294

    Article  CAS  PubMed  Google Scholar 

  21. Vanhecke D, Graber W, Studer D (2008) Chapter 9 close-to-native ultrastructural preservation by high pressure freezing. Methods Cell Biol 88:151–164

    Article  PubMed  Google Scholar 

  22. Valentijn KM, Valentijn JA, Jansen KA, Koster AJ (2008) A new look at Weibel-Palade body structure in endothelial cells using electron tomography. J Struct Biol 161(3):447

    Article  CAS  PubMed  Google Scholar 

  23. Berriman JA, Li S, Hewlett LJ, Wasilewski S, Kiskin FN, Carter T, Hannah MJ, Rosenthal PB (2009) Structural organization of Weibel-Palade bodies revealed by cryo-EM of vitrified endothelial cells. Proc Natl Acad Sci 106(41):17407–17412

    Article  PubMed  Google Scholar 

  24. van Nispen tot Pannerden H, de Haas F, Geerts W, Posthuma G, van Dijk S, Heijnen HF (2010) The platelet interior revisited: electron tomography reveals tubular alpha-granule subtypes. Blood 116(7):1147–1156

    Article  CAS  PubMed  Google Scholar 

  25. Valentijn KM, van Driel LF, Mourik MJ, Hendriks GJ, Arends TJ, Koster AJ, Valentijn JA (2010) Multigranular exocytosis of Weibel –Palade bodies in vascular endothelial cells. Blood 116(10):1807–1816

    Article  CAS  PubMed  Google Scholar 

  26. Mourik MJ, Faas FGA, Valentijn KM, Valentijn JA, Eikenboom JC, Koster AJ (2014) Correlative light microscopy and electron tomography to study van Willebrand factor exocytosis from vascular endothelial cells. Methods Cell Biol 124:71–92

    Article  PubMed  Google Scholar 

  27. Agronskaia AV, Valentijn JA, van Driel LF, Schneijdenberg CTWM, Humbel BM, van Bergen en Henegouwen PMP, Verkleij AJ, Koster AJ, Gerritsen HC (2008) Integrated fluorescence and transmission electron microscopy. J Struct Biol 164(2):183–189

    Article  CAS  PubMed  Google Scholar 

  28. Faas FGA, Bárcena M, Agronskaia AV, Gerritsen HC, Moscicka KB, Diebolder CA, van Driel LF, Limpens RWAL, Bos E, Ravelli RBG, Koning RI, Koster AJ (2013) Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy. J Struct Biol 181(3):283–290

    Article  CAS  PubMed  Google Scholar 

  29. de Boer P, Hoogenboom JP, Giepmans BNG (2015) Correlated light and electron microscopy: ultrastructure lights up! Nat Methods 12(6):503–513

    Article  CAS  PubMed  Google Scholar 

  30. Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116(1):71

    Article  CAS  PubMed  Google Scholar 

  31. van Donselaar EG, Posthuma G, Zeuschner D, Humbel BM, Slot JW (2007) Immunogold labeling of cryosections from high-pressure frozen cells. Traffic 8(5):471–485

    Article  CAS  PubMed  Google Scholar 

  32. Al-Amoudi A, Chang JJ, Leforestier A, McDowall A, Salamin LM, Norlén LP, Richter K, Blanc NS, Studer D, Dubochet J (2004) Cryo-electron microscopy of vitreous sections. EMBO J 23(18):3583–3588

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Schaffer M, Engel BD, Laugks T, Mahamid J, Plitzko JM, Baumeister W (2015) Cryo-focused ion beam sample preparation for imaging vitreous cells by cryo-electron tomography. Bio Protoc 5(17):e1575

    Article  PubMed  PubMed Central  Google Scholar 

  34. Heijnen HF, Oorschot V, Sixma JJ, Slot JW, James DE (1997) Thrombin stimulates glucose transport in human platelets via the translocation of the glucose transporter GLUT-3 from alpha-granules to the cell surface. J Cell Biol 138(2):323–330

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Heijnen HF, Debili N, Vainchencker W, Breton-Gorius J, Geuze HJ, Sixma JJ (1998) Multivesicular bodies are an intermediate stage in the formation of platelet alpha-granules. Blood 91(7):2313–2325

    PubMed  CAS  Google Scholar 

  36. Hoppe W (1974) Towards three-dimensional "electron microscopy" at atomic resolution. Naturwissenschaften 61(6):239–249

    Article  CAS  PubMed  Google Scholar 

  37. Baumeister W, Grimm R, Walz J (1999) Electron tomography of molecules and cells. Trends Cell Biol 9(2):81–85

    Article  CAS  PubMed  Google Scholar 

  38. Diebolder CA, Koster AJ, Koning RI (2012) Pushing the resolution limits in cryo electron tomography of biological structures. J Microsc 248(1):1–5

    Article  CAS  PubMed  Google Scholar 

  39. Gilbert PFC (1972) The reconstruction of a three-dimensional structure from projections and its application to electron microscopy. II direct methods. Proc R Soc London B Ser Biol Sci 182:89–102

    Article  CAS  Google Scholar 

  40. Kühlbrandt W (2014) The resolution revolution. Science 343(6178):1443–1444

    Article  PubMed  Google Scholar 

  41. Karreman MA, van Donselaar EG, Gerritsen HC, Verrips CT, Verkleij AJ (2011) VIS2FIX: a high-speed fixation method for immuno-electron microscopy. Traffic 12(7):806–814

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands

    Kasia B. Engberts & Willie J. C. Geerts

  2. Laboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, The Netherlands

    Cor Seinen & Harry F. G. Heijnen

  3. Department of Cell Biology, Cell Microscopy Core, University Medical Center Utrecht, Utrecht, The Netherlands

    Harry F. G. Heijnen

Authors
  1. Kasia B. Engberts
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  2. Cor Seinen
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  3. Willie J. C. Geerts
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  4. Harry F. G. Heijnen
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Corresponding author

Correspondence to Harry F. G. Heijnen .

Editor information

Editors and Affiliations

  1. Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Reading, Berkshire, UK

    Jonathan M. Gibbins

  2. Department of Molecular and Cell Biology, University of Leicester, Leicester, UK

    Martyn Mahaut-Smith

1 Electronic Supplementary Material

The movie shows the subsequent analytical steps for RT-CLEM and tomography of platelet whole mounts using the iCorrtm. Platelets spread conjugated anti-vWF and 10 nm Protein A gold. Regions of interest (green IF spots, reflecting luminal vWF release) were sequentially imaged in IF and TEM mode using the iCorr software package (iCorrtm FEI Company). Tilt series were recorded and aligned using the IMOD softwareon fibrinogen-coated EM supports were double immunolabeled with Alexa 488 (MP4 29686 kb)

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Engberts, K.B., Seinen, C., Geerts, W.J.C., Heijnen, H.F.G. (2018). Electron Tomography and Correlative Approaches in Platelet Studies. In: Gibbins, J., Mahaut-Smith, M. (eds) Platelets and Megakaryocytes . Methods in Molecular Biology, vol 1812. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8585-2_4

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  • DOI: https://doi.org/10.1007/978-1-4939-8585-2_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8584-5

  • Online ISBN: 978-1-4939-8585-2

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