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
Part of the Methods in Molecular Biology book series (MIMB, volume 1929)


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 


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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

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