Advertisement

Analysis of Ca2+-Dependent Weibel–Palade Body Tethering by Live Cell TIRF Microscopy: Involvement of a Munc13-4/S100A10/Annexin A2 Complex

  • Nina Criado Santos
  • Tarek Chehab
  • Anna Holthenrich
  • Volker GerkeEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)

Abstract

Endothelial cells respond to blood vessel injury by the acute release of the procoagulant von Willebrand factor, which is stored in unique secretory granules called Weibel–Palade bodies (WPBs). Stimulated, Ca2+-dependent exocytosis of WPBs critically depends on their proper targeting to the plasma membrane, but the mechanism of WPB-plasma membrane tethering prior to fusion is not well characterized. Here we describe a method to visualize and analyze WPB tethering and fusion in living human umbilical vein endothelial cells (HUVEC) by total internal reflection fluorescence (TIRF) microscopy. This method is based on automated object detection and allowed us to identify components of the tethering complex of WPBs and to monitor their dynamics in space and time. An important tethering factor identified by this means was Munc13-4 that was shown to interact with S100A10 residing in a complex with plasma membrane-bound annexin A2.

Key words

Annexin Calcium Endothelial cell Exocytosis Secretion S100 protein 

References

  1. 1.
    Weibel ER, Palade GE (1964) New cytoplasmic components in arterial endothelia. J Cell Biol 23:101–112CrossRefGoogle Scholar
  2. 2.
    Sadler JE (1998) Biochemistry and genetics of von Willebrand factor. Annu Rev Biochem 67:395–424.  https://doi.org/10.1146/annurev.biochem.67.1.395CrossRefPubMedGoogle Scholar
  3. 3.
    Wagner DD, Frenette PS (2008) The vessel wall and its interactions. Blood 111:5271–5281.  https://doi.org/10.1182/blood-2008-01-078204CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Nightingale T, Cutler D (2013) The secretion of von Willebrand factor from endothelial cells; an increasingly complicated story. J Thromb Haemost 11(Suppl. 1):192–201.  https://doi.org/10.1111/jth.12225CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Valentijn KM, Eikenboom J (2013) Weibel-Palade bodies: a window to von Willebrand disease. J Thromb Haemost 11:581–592.  https://doi.org/10.1111/jth.12160CrossRefPubMedGoogle Scholar
  6. 6.
    Matsushita K, Morrell CN, Cambien B et al (2003) Nitric oxide regulates exocytosis by S-nitrosylation of N-ethylmaleimide-sensitive factor. Cell 115:139–150CrossRefGoogle Scholar
  7. 7.
    Pulido IR, Jahn R, Gerke V (2011) VAMP3 is associated with endothelial Weibel-Palade bodies and participates in their Ca(2+)-dependent exocytosis. Biochim Biophys Acta 1813:1038–1044.  https://doi.org/10.1016/j.bbamcr.2010.11.007CrossRefPubMedGoogle Scholar
  8. 8.
    van Breevoort D, van Agtmaal EL, Dragt BS et al (2012) Proteomic screen identifies IGFBP7 as a novel component of endothelial cell-specific Weibel-Palade bodies. J Proteome Res 11:2925–2936.  https://doi.org/10.1021/pr300010rCrossRefPubMedGoogle Scholar
  9. 9.
    van Breevoort D, Snijders AP, Hellen N et al (2014) STXBP1 promotes Weibel-Palade body exocytosis through its interaction with the Rab27A effector Slp4-a. Blood 123:3185–3194.  https://doi.org/10.1182/blood-2013-10-535831CrossRefPubMedGoogle Scholar
  10. 10.
    Fish KN (2009) Total internal reflection fluorescence (TIRF) microscopy. Curr Protoc Cytom 12:Unit12.18. doi:  https://doi.org/10.1002/0471142956.cy1218s50
  11. 11.
    Schuberth CE, Tängemo C, Coneva C et al (2015) Self-organization of core Golgi material is independent of COPII-mediated endoplasmic reticulum export. J Cell Sci 128:1279–1293.  https://doi.org/10.1242/jcs.154443CrossRefPubMedGoogle Scholar
  12. 12.
    Chehab T, Santos NC, Holthenrich A et al (2017) A novel Munc13-4/S100A10/annexin A2 complex promotes Weibel-Palade body exocytosis in endothelial cells. Mol Biol Cell 28:1688–1700.  https://doi.org/10.1091/mbc.E17-02-0128CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Neeft M, Wieffer M, de Jong AS et al (2005) Munc13-4 is an effector of Rab27a and controls secretion of lysosomes in hematopoietic cells. Mol Biol Cell 16:731–741.  https://doi.org/10.1091/mbc.E04-10-0923CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Boswell KL, James DJ, Esquibel JM et al (2012) Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion. J Cell Biol 197:301–312.  https://doi.org/10.1083/jcb.201109132CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Babich V, Meli A, Knipe L et al (2008) Selective release of molecules from Weibel-Palade bodies during a lingering kiss. Blood 111:5282–5290.  https://doi.org/10.1182/blood-2007-09-113746CrossRefPubMedGoogle Scholar
  16. 16.
    Jaffe EA, Nachman RL, Becker CG, Minick CR (1973) Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52:2745–2756.  https://doi.org/10.1172/JCI107470CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Harrison-Lavoie KJ, Michaux G, Hewlett L et al (2006) P-selectin and CD63 use different mechanisms for delivery to Weibel-Palade bodies. Traffic 7:647–662.  https://doi.org/10.1111/j.1600-0854.2006.00415.xCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Nina Criado Santos
    • 1
    • 2
  • Tarek Chehab
    • 1
  • Anna Holthenrich
    • 1
  • Volker Gerke
    • 1
    Email author
  1. 1.Centre for Molecular Biology of Inflammation, Institute of Medical Biochemistry, University of MünsterMünsterGermany
  2. 2.Department of Cell Physiology and MetabolismUniversity of GenevaGenevaSwitzerland

Personalised recommendations