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MBC in Press, published online ahead of print July 11, 2003
Mol. Biol. Cell 10.1091/mbc.E02-11-0726

A more recent version of this article appeared on October 1, 2003
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Submitted on November 12, 2002
Revised on May 13, 2003
Accepted on May 30, 2003

{beta}cap73-ARF6 Interactions Modulate Cell Shape and Motility Following Injury in vitro

KN Riley, AE Maldonado, P Tellier, C D'Souza-Schorey1, and IM Herman*

1 Biol Dept, Univ Notre Dame, Notre Dame, IN

* Corresponding author. E-mail address: ira.herman{at}tufts.edu.

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 {clubsuit}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, {beta}cap73. Coimmunoprecipitation and Western blot analyses reveal the direct association of ARF6-HA with {beta}cap73, 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.






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