Defining the Role of Protein Interactions at WRAMP Structures in Directional Migration

Abstract

Cell migration is a critical process in development, tissue regeneration, and cancer cell invasion, but mechanisms that establish the location of the cell rear and activate rear contractile forces are still poorly understood. Our lab previously discovered a protein network named the “Wnt5a‐receptor‐actomyosin‐polarity (WRAMP) structure” which may serve as a novel mechanism for rear‐directed control of migration. The WRAMP structure forms transiently in response to the signaling ligand Wnt5a and is characterized by polarization of F‐actin, myosin‐IIB, and melanoma cell adhesion molecule (MCAM). Cells with WRAMP structures migrate in a persistent direction for longer periods of time compared to cells without WRAMP structures, and the location where WRAMP structures form may determine the position of the cell rear and direction of migration. However, the specific interactions of proteins at WRAMP structures that activate actomyosin contraction are unknown. To address this question, we have generated plasmids to express previously identified WRAMP structure members MCAM and moesin fused to the engineered peroxidase APEX2 or the promiscuous biotin ligase BirA(R118G) for use in the proteomics techniques APEX and BioID, respectively. In these methods, proteins in close proximity to the bait protein are biotinylated and subsequently isolated on streptavidin beads for identification by mass spectrometry. Ongoing goals of this project are to compare APEX and BioID methods in melanoma cells to build up a WRAMP structure “interactome,” prioritized by interactions that increase in the presence of Wnt5a treatment. In addition, we are examining the role of RhoA/Rho kinase (ROCK) signaling in activation of actomyosin contraction using immunofluorescence for activated substrates of ROCK at WRAMP structures, and we will test a literature‐based model for RhoA activation through MCAM‐moesin interactions using an MCAM mutant which lacks moesin binding. These findings will help define the spatial organization of WRAMP structures and identify key interactions and signaling pathways involved in control of cell migration.Support or Funding InformationThis work is supported by funding from the National Institutes of Health (R01GM105997 and T32GM008759).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.