Betacap73-ARF6 interactions modulate cell shape and motility after injury in vitro.
To understand the role that ARF6 plays in regulating isoactin dynamics and cell motility, we transfected endothelial cells (EC) with HA-tagged ARF6: the wild-type form (WT), a constitutively-active form unable to hydrolyze GTP (Q67L), and two dominant-negative forms, which are either unable to release GDP (T27N) or fail to bind nucleotide (N122I). Motility was assessed by digital imaging microscopy before Western blot analysis, coimmunoprecipitation, or colocalization studies using ARF6, beta-actin, or beta-actin-binding protein-specific antibodies. EC expressing ARF6-Q67L spread and close in vitro wounds at twice the control rates. EC expressing dominant-negative ARF6 fail to develop a leading edge, are unable to ruffle their membranes (N122I), and possess arborized processes. Colocalization studies reveal that the Q67L and WT ARF6-HA are enriched at the leading edge with beta-actin; but T27N and N122I ARF6-HA are localized on endosomes together with the beta-actin capping protein, betacap73. Coimmunoprecipitation and Western blot analyses reveal the direct association of ARF6-HA with betacap73, defining a role for ARF6 in signaling cytoskeletal remodeling during motility. Knowledge of the role that ARF6 plays in orchestrating membrane and beta-actin dynamics will help to reveal molecular mechanisms regulating actin-based motility during development and disease.
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000199:Actins / Q000378:metabolism* / Q000502:physiology; D000818:Animals; D002417:Cattle; D002465:Cell Movement / Q000502:physiology*; D048429:Cell Size / Q000502:physiology; D022081:Cell Surface Extensions / Q000378:metabolism / Q000502:physiology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D042783:Endothelial Cells; D006153:Guanosine Diphosphate / Q000235:genetics / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins / Q000378:metabolism* / Q000502:physiology; D018715:Microscopy, Video; D009154:Mutation; D012160:Retina / Q000378:metabolism* / Q000502:physiology; D014947:Wounds and Injuries
D000089982:ADP-Ribosylation Factor 6; D000199:Actins; D008840:Microfilament Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020727:ADP-Ribosylation Factors