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

Von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized and secreted by endothelial cells and stored in Weibel-Palade bodies (WPBs). The secretory Rab GTPases Rab27A, Rab3B and Rab3D have been linked with WPB trafficking and secretion. How these Rabs are activated and recruited to WPBs remains elusive. In this study, we identified MAP kinase-activating death domain (MADD) as the guanine nucleotide exchange factor (GEF) for Rab27A and both Rab3 isoforms in primary human endothelial cells. Rab activity assays revealed a reduction in Rab27A, Rab3D, and Rab3B activation upon MADD silencing. Rab activation, but not binding, was dependent on the DENN domain of MADD, indicating potential existence of two Rab interaction modules. Furthermore, immunofluorescent analysis showed that Rab27A, Rab3B, and Rab3D recruitment to WPBs was dramatically decreased upon MADD knockdown, revealing that MADD drives Rab membrane targeting. Artificial mistargeting of MADD using a TOMM70-tag abolished Rab27A localization to WPB membranes in a DENN domain-dependent manner, indicating that normal MADD localization in the cytosol is crucial. Activation of Rab3B and Rab3D was reduced upon Rab27A silencing, suggesting that activation of these Rabs is enhanced through prior activation of Rab27A by MADD. MADD silencing did not affect WPB morphology, but reduced VWF intracellular content. Furthermore, MADD-depleted cells exhibited decreased histamine-evoked VWF release, similar to Rab27A-depleted cells. In conclusion, MADD acts as a master regulator in VWF secretion by coordinating the activation and membrane targeting of secretory Rabs to WPBs.

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

Cited by 3 publications

References 17 publications

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GDP/GTP exchange factor MADD drives activation and recruitment of secretory Rab GTPases to Weibel-Palade bodies

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