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α₆β₄ integrin is localized in a unique punctate distribution at the cell–substratum interface along the leading front of single, front–rear–polarized A549 cells. These puncta are interspersed between focal adhesions and lack association with the actin cytoskeleton. Knockdown of β₄ integrin in A549 cells inhibits their directed migration, with knockdown cells exhibiting large focal adhesions and reduced actin dynamics. Despite these changes, the speed of knockdown cells is equivalent to control cells. Interestingly, in such cells, α₆ integrin retains its punctate distribution. Moreover, in β₄ integrin knockdown cells, we observe a loss of β₁ integrin from focal adhesions and an enhanced association with α₆ integrin. We confirmed the switch in the β integrin binding partner of α₆ integrin in the knockdown cells by immunoprecipitation. We next investigated the role of β₄ integrin in collective cell migration. Wounded …