EBP50, also known as NHERF1, is a scaffold protein that possesses two PDZ interacting domains and an ERM binding domain. In a previous study, we characterized the interaction of EBP50 with several proteins of the cell cytoskeleton in intact arteries, after agonist stimulation. The aim of this work was to further characterize the role of EBP50 in the modulation of cell cytoskeleton in primary vascular smooth muscle cells.

Immunostaining of EBP50 in these cells revealed a localization along thick filaments that crosses the entire cell. Transfection of two different siRNAs against EBP50 depleted its expression by 99±0.5% and 97±0.5% after 96 hours of culture. Cells transfected with a scramble siRNA exhibited thick actin bundles, large and numerous focal adhesions and microtubules that spread uniformly in the cell from the perinuclear region. On the other hand, cells depleted for EBP50 lack actin bundles, show lamellipodiae and trailing tails, had fewer and smaller focal adhesions and microtubules mainly spread into lamellae projections. Cells transfected with a scramble siRNA were able to migrate constitutively. EBP50-knocked down cells exhibited faster migratory properties. Moreover, 19±0.5 and 21±1% of binucleated cells were observed in cells transfected with first and second siRNAs against EBP50, respectively, while only 5±1% of binucleated cells were observed among cells transfected with a scramble siRNA. This observation has been confirmed by propidium iodure staining and FACS analysis of DNA content: 6±0.5% and 16±2% of the cells were in G2/M phase, respectively in control cells and in cells transfected with EBP50 specific siRNA, indicating an alteration in the cytokinesis. These results indicate that EBP50 is a key player in cellular migration and cytokinesis. Further studies would be performed to investigate how EBP50 modulates these cellular processes.