Whether ␣6␤4 integrin regulates migration remains controversial. ␤4 integrin-deficient (JEB) keratinocytes display aberrant migration in that they move in circles, a behavior that mirrors the circular arrays of laminin (LM)-332 in their matrix. In contrast, wild-type keratinocytes and JEB keratinocytes, induced to express ␤4 integrin, assemble laminin-332 in linear tracks over which they migrate. Moreover, laminin-332-dependent migration of JEB keratinocytes along linear tracks is restored when cells are plated on wild-type keratinocyte matrix, whereas wild-type keratinocytes show rotation over circular arrays of laminn-332 in JEB keratinocyte matrix. The activities of Rac1 and the actin cytoskeleton-severing protein cofilin are low in JEB keratinocytes compared with wild-type cells but are rescued following expression of wild-type ␤4 integrin in JEB cells. Additionally, in wild-type keratinocytes Rac1 is complexed with ␣6␤4 integrin. Moreover, Rac1 or cofilin inactivation induces wild-type keratinocytes to move in circles over rings of laminin-332 in their matrix. Together these data indicate that laminin-332 matrix organization is determined by the ␣6␤4 integrin/actin cytoskeleton via Rac1/cofilin signaling. Furthermore, our results imply that the organizational state of laminin-332 is a key determinant of the motility behavior of keratinocytes, an essential element of skin wound healing and the successful invasion of epidermal-derived tumor cells.Migration of cells is an essential element of morphogenesis, remodeling following tissue damage and metastasis. For example, following wounding of the skin, epidermal cells migrate over a wound, a complex process involving changes in cytoskeleton organization, alterations in cell-cell and cell-matrix interactions, and modulation in gene and protein expression (1, 2).In particular, keratinocytes lose stable matrix adhesive structures called hemidesmosomes, migrate over exposed dermal collagen, and deposit a provisional matrix rich in laminin (LM) 3 -332 (old nomenclature: laminin-5), a heterotrimer consisting of ␣3, ␤3, and ␥2 subunits (1-5). This provisional matrix regulates the motility of keratinocytes (2-4). LM-332 is also believed to support tumor cell invasion (6 -12).Although cells in wounds and cancer cells migrate on a LM-332 substrate, LM-332 stabilizes epidermal cell adhesion in intact skin (3, 4,(12)(13)(14)(15)(16). These contradictory functions exhibited by LM-332, adhesion and motility, have been investigated by a number of laboratories, and results to date indicate that proteolytic processing of LM-332 has a profound impact on its functions. Specifically, in the extracellular matrix, the ␣3 and ␥2 subunits are processed from 190 to 160 kDa and from 140 to 100 kDa, respectively (4, 17-22). LM-332 containing an unprocessed ␣3 chain supports keratinocyte motility (20). Upon proteolytic cleavage of the ␣3 LM subunit within its G domain, LM-332 triggers hemidesmosome assembly, leading to decreased cell motility (3, 20 -24). However, further proteolysis of l...

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