In vivo and in vitro analyses of the functional role of alpha4 integrin in cell migration

The α4β1 integrin is known to play an important role in cell migration both in vivo and in vitro. The mechanism by which α4β1 promotes cell migration is likely to be different from those integrins found in focal adhesions, such as α5β1, as it is not localized to focal adhesions in most cell types and has different ligand-binding specificities. While α4β1 is known to play an important role in cell migration, questions remain concerning its exact role. By examining the early stages of mouse embryogenesis by RNA in situ hybridization and immunohistochemical analyses, we found that α4 integrin is expressed in two early migratory cell populations between embryonic days 8 and 10.5. α4 expression was observed in cranial neural crest cells and in epicardial progenitor cells, implicating a role for α4 integrin during cell migration in cardiac development. Using in vitro studies, we sought to understand the mechanism by which α4β1 promotes cell migration. When ectopically expressed in Chinese hamster ovary cells, α4β1, but not α5β1, is sufficient and required for the generation of broad lamellipodia in response to scratch-wounding. Cells expressing an α4/GFP fusion protein formed transient puncta at the leading edge of protruding lamellipodia in response to a scratch-wound assay. Cells expressing a mutant form of α4/GFP which disrupts paxillin binding exhibited earlier formation of broad lamellipodia and α4-positive puncta. Although this mutation enhanced lamellipodia formation, random motility was reduced relative to that of cells expressing wild-type α4 indicating that lamellipodia protrusion and random motility are distinct types of motile activities differentially regulated by α4/paxillin interactions. While α4-positive puncta did not contain paxillin, α4β1 and paxillin did colocalize in ruffles. These results show that α4β1 may be promoting the formation of broad lamellipodia by forming transient clusters at the leading edge and that paxillin may be regulating their formation. Our studies have shown that α4 integrin is expressed at key cell migration events during early mouse development. Furthermore, we have provided evidence that this migration is likely to be promoted via a mechanism distinct from that of integrins which promote cell motility via focal complex/focal adhesion involvement.