Abstract
ABSTRACTWeibel–Palade body (WPB)–actin interactions are essential for the trafficking and secretion of von Willebrand factor; however, the molecular basis for this interaction remains poorly defined. Myosin Va (MyoVa or MYO5A) is recruited to WPBs by a Rab27A–MyRIP complex and is thought to be the prime mediator of actin binding, but direct MyRIP–actin interactions can also occur. To evaluate the specific contribution of MyRIP–actin and MyRIP–MyoVa binding in WPB trafficking and Ca2+-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-binding rather than its MyoVa-binding properties. We also show that, although the role of MyRIP in Ca2+-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.
Highlights
Weibel–Palade bodies (WPBs) are endothelial cell secretory granules containing the adhesive glycoprotein von Willebrand factor (VWF)
We demonstrate that the role of MyRIP can be only partly accounted for by its recruitment of MyoVa, and that MyRIP–actin interactions play a prominent role for both trafficking and exocytosis
Ca2+-driven WPB exocytosis after actin disruption or stabilisation To directly assess the effect of actin disruption on the kinetics of Ca2+-driven WPB exocytosis, we used live-cell imaging of human umbilical vein endothelial cells (HUVECs) expressing the WPBspecific luminal marker EGFP-tagged VWF propolypeptide (VWFpp–EGFP) (Hannah et al, 2005), which does not directly interfere with interactions between WPBs and cytoskeletal components or plasma membrane during exocytosis
Summary
Weibel–Palade bodies (WPBs) are endothelial cell secretory granules containing the adhesive glycoprotein von Willebrand factor (VWF). Mutations in VWF that result in a qualitative or quantitative defect in function cause a family of bleeding disorders collectively called von Willebrand disease (Sadler et al, 2006). Molecular defects affecting the trafficking or exocytosis of WPBs can give rise to alterations in circulating VWF and cause bleeding disorders (van Loon et al, 2010; Zhu et al, 2014).
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