Metastasis suppressor 1 interacts with α-actinin 4 to affect its localization and regulate formation of membrane ruffling

Membrane ruffling plays an important role in the directed cell migration and escape of tumor cells from the monolayer. Metastasis suppressor 1 (MTSS1), also known as missing in metastasis, has been implicated in cell morphology, motility, metastasis, and development. Here, the dynamic interaction proteins associated with MTSS1 and involved in membrane ruffling were determined by cross-linking and mass spectrometry analysis. We identified α-actinin 4 (ACTN4) as an interacting protein and confirmed a direct interaction between MTSS1 and ACTN4. Moreover, co-expression of MTSS1 in fibroblasts recruited cytoplasmic ACTN4 to the cell periphery, at which point ruffling became thick and rigid. In MCF-7 cells, MTSS1 knockdown did not show an obvious effect on the cell shape or the distribution of endogenous ACTN4; however, ACTN4 overexpression transformed cell morphology from an epidermal- to a fibroblast-like shape, and further MTSS1 depletion significantly increased the ratio of fibroblast cells exhibiting prominent ruffling. Furthermore, biochemical data suggested that MTSS1 cross-linking with ACTN4 induced the formation of actin fiber bundles into more organized structures in vitro. These data indicated that MTSS1 might recruit cytoplasmic ACTN4 to the cell periphery and regulate cytoskeleton dynamics to restrict its performance in membrane ruffling.