10779/crick.12652940.v1 Ianina L Conte Ianina L Conte Nicola Hellen Nicola Hellen Ruben Bierings Ruben Bierings Gregory I Mashanov Gregory I Mashanov Jean-Baptiste Manneville Jean-Baptiste Manneville Nikolai I Kiskin Nikolai I Kiskin Matthew J Hannah Matthew J Hannah Justin E Molloy Justin E Molloy Tom Carter Tom Carter Interaction between MyRIP and the actin cytoskeleton regulates Weibel-Palade body trafficking and exocytosis The Francis Crick Institute 2020 Actin Exocytosis MyRIP Myosin Va Weibel–Palade body Actin Cytoskeleton Actins Calcium Cell Line Cell Movement Green Fluorescent Proteins Human Umbilical Vein Endothelial Cells Humans Myosin Heavy Chains Myosin Type V Protein Binding Protein Transport Vesicular Transport Proteins Weibel-Palade Bodies Molloy FC001119 Schaefer FC001153 06 Biological Sciences 11 Medical and Health Sciences Developmental Biology 2020-07-15 11:15:21 Journal contribution https://crick.figshare.com/articles/journal_contribution/Interaction_between_MyRIP_and_the_actin_cytoskeleton_regulates_Weibel-Palade_body_trafficking_and_exocytosis/12652940 Weibel-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 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-binding rather than its 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.