Abstract

Weibel-Palade body (WPB)-actin interactions are essential for the trafficking and secretion of von Willebrand factor, yet, the molecular basis for this interaction remains poorly defined. Myosin Va (MyoVa) is recruited to WPBs by a Rab27A-MyRIP complex and is thought to be the prime mediator of actin binding, however, direct MyRIP-actin interactions can also occur. To evaluate the specific contribution of MyRIP-actin and MyRIP-MyoVa binding in WPB trafficking and Ca(2+)-driven exocytosis we used EGFP-MyRIP point mutants with disrupted MyoVa and/or actin binding and high-speed live-cell fluorescence microscopy. We now show that the ability of MyRIP to restrict WPB movement depends upon its actin rather than MyoVa binding properties. We also show that although the role of MyRIP in Ca(2+)-driven exocytosis requires both MyoVa and actin binding potential, it is the latter that plays a dominant role. In view of these results and together with the analysis of actin disruption or stabilisation experiments we propose that the role of MyRIP in regulating WPB trafficking and exocytosis is mediated largely through its interaction with actin rather than with MyoVa.