Filamin A-Hinge Region 1-EGFP: A Novel Tool for Tracking the Cellular Functions of Filamin A in Real-Time

<div><h3>Abstract</h3><h3>Background</h3><p>Filamin A (FLNa) is an actin-crosslinking protein necessary for stabilizing the cell surface, organizing protrusive activity and for promoting efficient cellular translocation. Recently, our group demonstrated the requirement of FLNa for the internalization of the chemokine receptor CCR2B.</p> <h3>Methodology and Principal Findings</h3><p>In order to study the role of FLNa <em>in vitro</em> and in real-time, we have developed a fluorescent FLNa-EGFP construct. In this novel imaging tool, we introduced the EGFP-tag inside the flexible hinge 1 region of FLNa between two calpain cleavage sites. Our findings indicate that the FLNa-EGFP construct was correctly expressed, cleaved by calpain and colocalized with actin filaments as shown by immunostaining experiments in the human melanoma cell lines A7 (FLNa-repleted) and M2 (FLNa-deficient). In addition, scanning-electron microscopy (SEM) and micropatterning studies also provided clear evidence that the cell rigidity was restored. FLNa-EGFP allowed us to demonstrate the interaction of FLNa with the chemokine receptor CCR2B in endocytic vesicles after CCL2 ligand stimulation. Through live-cell imaging studies we show that the CCR2B receptor in Rab5-positive vesicles moves along filamin A-positive fibers.</p> <h3>Significance</h3><p>Taken together, these results outline the functionality of the FLNa-EGFP and the importance of filamin A for receptor internalization and movement into endocytic vesicles.</p>