Abstract
Cell migration is a complex process that is coordinately regulated by cell-matrix adhesion and actin cytoskeleton. We report here that migfilin, a recently identified component of cell-matrix adhesions, is a biphasic regulator of cell migration. Loss of migfilin impairs cell migration. Surprisingly, overexpression of migfilin also reduces cell migration. Molecularly, we have identified vasodilator-stimulated phosphoprotein (VASP) as a new migfilin-binding protein. The interaction is mediated by the VASP EVH1 domain and a single L104PPPPP site located within the migfilin proline-rich domain. Migfilin and VASP form a complex in both suspended and adhered cells, and in the latter, they co-localize in cell-matrix adhesions. Functionally, migfilin facilitates VASP localization to cell-matrix adhesions. Using two different approaches (VASP-binding defective migfilin mutants and small interfering RNA-mediated VASP knockdown), we show that the interaction with VASP is crucially involved in migfilin-mediated regulation of cell migration. Our results identify migfilin as an important regulator of cell migration and provide new information on the mechanism by which migfilin regulates this process.
Highlights
Migfilin is a recently identified widely expressed focal adhesion protein that provides a link between cell-matrix adhesions and the actin cytoskeleton [5]
Using two different approaches, we show that the interaction with vasodilator-stimulated phosphoprotein (VASP) is crucially involved in migfilin regulation of cell migration
FLAGmigfilin was immunoprecipitated with anti-FLAG mAb M2 (Fig. 1A, lane 4) and the immunoprecipitates were probed by Western blotting with an anti-VASP Ab
Summary
Antibodies—The following primary Abs were used in this study: mouse anti-VASP mAb (BD Transduction Laboratories), rabbit polyclonal anti-VASP Ab (Calbiochem), rabbit polyclonal anti-focal adhesion kinase Ab (Santa Cruz), rabbit anti-GFP Ab (Santa Cruz), mouse anti-migfilin mAb (clone 43) [5], mouse anti-FLAG mAb M2 (Sigma), and mouse anti-vinculin mAb (Sigma). Yeast Two-hybrid Assays—A cDNA fragment encoding human migfilin residues 1–189 was inserted into the pGBKT7 vector (Clontech). The construct was used as bait to screen a human keratinocyte MATCHMAKER cDNA library following the manufacturer’s protocol (Clontech). To identify the VASP domain that mediates migfilin binding, cDNA fragments encoding human VASP sequences were inserted into the pGADT7 vector. The cDNA fragments encoding migfilin sequences were inserted into the pGBKT7 vector. The pGADT7 and pGBKT7 vectors containing VASP or migfilin sequences were introduced into yeast cells (Saccharomyces cerevisiae strain AH109) and the interaction was analyzed following the manufacturer’s protocol (Clontech). DNA Constructs, Transfection, and Immunoprecipitation—DNA vectors encoding FLAG-migfilin or FLAG-migfilin(s) were described [5, 12]. To generate the vector encoding the GFP-tagged migfilin proline-rich domain, a cDNA fragment encoding migfilin residues 84–180 was ligated into the pEGFP-C2 vector (Clontech).
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