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Calcium-Binding Proteins of the EF-Hand Superfamily pp 437–445Cite as

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

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Part of the book series: Methods in Molecular Biology ((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.

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Author notes

  1. Nina Criado Santos

    Present address: Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland

Authors and Affiliations

  1. Centre for Molecular Biology of Inflammation, Institute of Medical Biochemistry, University of Münster, Münster, Germany

    Nina Criado Santos, Tarek Chehab, Anna Holthenrich & Volker Gerke

Authors
  1. Nina Criado Santos
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  2. Tarek Chehab
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  3. Anna Holthenrich
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  4. Volker Gerke
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Corresponding author

Correspondence to Volker Gerke .

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Editors and Affiliations

  1. Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zürich, Zürich, Switzerland

    Claus W. Heizmann

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Criado Santos, N., Chehab, T., Holthenrich, A., Gerke, V. (2019). Analysis of Ca2+-Dependent Weibel–Palade Body Tethering by Live Cell TIRF Microscopy: Involvement of a Munc13-4/S100A10/Annexin A2 Complex. In: Heizmann, C. (eds) Calcium-Binding Proteins of the EF-Hand Superfamily. Methods in Molecular Biology, vol 1929. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9030-6_27

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  • DOI: https://doi.org/10.1007/978-1-4939-9030-6_27

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9029-0

  • Online ISBN: 978-1-4939-9030-6

  • eBook Packages: Springer Protocols

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