Invadopodia are actin-rich membrane protrusions which play essential roles in cancer cell invasiveness [1]. Ezrin and radixin, closely related F-actin-plasma membrane linkers of the ERM protein family, have both been implicated in metastasis, while the more distantly related protein merlin functions as a tumor suppressor [2, 3]. The Na+/H+ exchanger NHE1 localizes to invadopodia and is implicated in regulation of cancer cell motility [4, 5]. NHE1 interacts directly with ERM proteins, however, the possible link between NHE1 and ERM proteins in regulation of invasiveness and invadopodia function, and the possible functional relation between NHE1 and merlin are both unelucidated. The aim of this project was to characterize the roles and interactions of NHE1, ezrin, radixin and merlin in regulation of invasiveness and growth in MCF-7 breast cancer cells. Experiments were carried out in the absence and presence of constitutively active, N-terminally truncated ErbB2 (ΔNErbB2), which we and others have shown increases motility and invasiveness of these cells [5]. Expression of ezrin, but not of radixin or merlin was upregulated in ΔNErbB2 expressing MCF-7 cells. Ezrin and radixin colocalized strongly with NHE1 in invadopodial rosettes both in the absence and presence of ΔNErbB2, while merlin did not localize to these structures. siRNA-mediated knockdown of NHE1 increased the average number of invadopodia per cell from 0.23 ± 0.040 to 0.30 ± 0.020 in vector cells (n=3), yet had no effect in ΔNErbB2 cells. Overexpression of wild-type, constitutively active or dominant negative ezrin significantly decreased the average number of invadopodia per cell from 0.30 ± 0.042 to 0.21 ± 0.047, 0.12 ± 0.020 or 0.10 ± 0.017 in vector cells respectively (n=3), whereas it again had no effect in ΔNErbB2 cells. The size of the invadopodia was unaffected by knockdown of NHE1 or knockdown or overexpression of ezrin. Ongoing Boyden chamber- and zymography experiments are evaluating the impact of merlin and NHE1 and/or ezrin knockdown on MCF-7 cell invasiveness. In accordance with its known role as a tumor suppressor, siRNA-mediated knockdown of merlin significantly increased the growth rate of MCF-7 cells, in a manner more pronounced in vector- than in ΔNErbB2-expressing cells. We speculate that this may reflect the dual roles of these proteins in control of invasiveness and proliferation, however, further studies are ongoing to determine the mechanisms involved. In conclusion, NHE1, ezrin, and radixin colocalized strongly in MCF-7 invadopodial rosettes whereas merlin was not found in these structures. Surprisingly, knockdown of not only merlin, but also of either NHE1, ezrin, or radixin, stimulated cell growth, in a manner most pronounced in the absence of ΔNErbB2 signaling.