Live-cell imaging and CLEM reveal the existence of ACTN4-dependent ruffle-edge lamellipodia acting as a novel mode of cell migration

α-Actinin-4 (ACTN4) expression levels are correlated with the invasive and metastatic potential of cancer cells; however, the underlying mechanism remains unclear. Here, we identified ACTN4-localized ruffle-edge lamellipodia using live-cell imaging and correlative light and electron microscopy (CLEM). BSC-1 cells expressing EGFP-ACTN4 showed that ACTN4 was most abundant in the leading edges of lamellipodia, although it was also present in stress fibers and focal adhesions. ACTN4 localization in lamellipodia was markedly diminished by phosphoinositide 3-kinase inhibition, whereas its localization in stress fibers and focal adhesions remained. Furthermore, overexpression of ACTN4, but not ACTN1, promoted lamellipodial formation. Live-cell analysis demonstrated that ACTN4-enriched lamellipodia are highly dynamic and associated with cell migration. CLEM revealed that ACTN4-enriched lamellipodia exhibit a characteristic morphology of multilayered ruffle-edges that differs from canonical flat lamellipodia. Similar ruffle-edge lamellipodia were observed in A549 and MDA-MB-231 invasive cancer cells. ACTN4 knockdown suppressed the formation of ruffle-edge lamellipodia and cell migration during wound healing in A549 monolayer cultures. Additionally, membrane-type 1 matrix metalloproteinase was observed in the membrane ruffles, suggesting that ruffle-edge lamellipodia have the ability to degrade the extracellular matrix and may contribute to active cell migration/invasion in certain cancer cell types.