Integrin α3β1 directs the stabilization of a polarized lamellipodium in epithelial cells through activation of Rac1

Abstract

Epithelial cell migration is a crucial event in wound healing, yet little is known about mechanisms whereby integrins regulate epithelial cell polarization and migration. In the present work, we demonstrate the importance of adhesion through the α3β1 integrin in promoting the stabilization of leading lamellipodia in migrating keratinocytes. We demonstrate that this integrin is found at the leading edge of migrating keratinocytes and that inhibition of α3β1 binding to laminin-5 prevents the formation of stable leading lamellipodia. Consistent with this observation, keratinocytes derived from α3β1-deficient mice fail to form stable leading lamellipodia but retain the ability to form actin-containing protrusions that rapidly extend and retract from the cell membrane. Formation of a leading lamellipodium also requires α3β1-dependent activation of Rac1, because α3β1-deficient keratinocytes show decreased activation of Rac1 compared with α3β1-expressing cells, and formation of stable leading lamellipodia can be inhibited in the latter cells by expression of the dominant negative Rac1 mutant Rac1N17. Furthermore, α3β1-deficient keratinocytes expressing constitutively active Rac1L61 failed to form stable lamellipodia when plated onto laminin-5, demonstrating that α3β1 is required for Rac1-mediated formation of a stable lamellipodium. These observations identify a crucial role for integrin-mediated adhesion and signaling in the formation of large, polarized, stable lamellipodia by migrating epithelial cells. To our knowledge, this study is the first to demonstrate that signal transduction through a specific integrin is required to direct the development of a lamellipodium from an initial protrusion and promote persistent epithelial cell migration.

Integrin α3β1 inhibits directional migration and wound re-epithelialization in the skin

“…Similarly, human keratinocytes defective in Ln-332 expression because of a deletion in the LAMB3 gene were found to migrate with decreased velocity and directional persistence (Hartwig et al, 2007). The apparent paradox is most likely explained by differences in the expression of laminin-binding integrins and the matrices used; in the absence of α6β1 integrin, α3β1 is required for polarization, cell spreading and migration (Choma et al, 2004), whereas on a different ligand or a complex matrix offering various ECM components (as in vivo when keratinocytes migrate over a provisionally formed matrix), migration is driven by other integrins whereas the laminin-binding integrins maintain adhesion to endogenous Ln-332 deposits at the rear of the cell. Interaction with these deposits regulates cell polarization (Frank and Carter, 2004), and is crucial to maintain adhesion during migration.…”

Laminin-5-integrin interaction signals through PI 3-kinase and Rac1b to promote assembly of adherens junctions in HT-29 cells

“…Bars, 25 m. vesicular trafficking (reviewed by Wymann and Pirola, 1998). Adhesion to laminin 5 through ␣ 3 ␤ 1 activates PI 3-kinase and regulates cell spreading and migration (Choma et al, 2004;Enserink et al, 2004). This effect could be modulated by ␣ 6 ␤ 4 adhesion (Nguyen et al, 2000a;Russell et al, 2003).…”

“…By contrast, cells adhering to fibronectin develop stress fibers and focal contacts by regulating integrin-dependent Rho activation. On laminin 5, ␣ 3 ␤ 1 -dependent activation of Rac1 contributes to the formation of stable lamellipodia necessary for cell migration (Choma et al, 2004). In keratinocytes, deposit of laminin 5 induces a change in signaling from a Rhoto a PI 3-kinase-dependent pathway (Nguyen et al, 2000a).…”

Single-molecule tracking of small GTPase Rac1 uncovers spatial regulation of membrane translocation and mechanism for polarized signaling

“…To examine whether the translocation of Rac1 to the membrane is a spatially regulated process, we carried out SPT measurements on MCF7 cells during cell spreading on collagen substrate, a condition that activates Rac1 (17,24,25). Isolated MCF7 cells were seeded onto glass coverslips coated with type I collagen to initiate cell spreading, and single molecules of Rac1 molecules were imaged during the process ( Fig.…”