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Vol. 15, Issue 1, 256-267, January 2004
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* Division of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037;
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037; and
|| Center for Oncology and Cell Biology, North Shore-Long Island Jewish Research Institute, Manhasset, New York 11030
Submitted January 17, 2003;
Revised September 3, 2003;
Accepted September 8, 2003
Monitoring Editor: Anne Ridley
The GTPase dynamin controls a variety of endocytic pathways, participates in the formation of phagosomes, podosomal adhesions, and invadopodia, and in regulation of the cytoskeleton and apoptosis. Rac, a member of the Rho family of small GTPases, controls formation of lamellipodia and focal complexes, which are critical in cell migration and phagocytosis. We now show that disruption of dynamin-2 function alters Rac localization and inhibits cell spreading and lamellipodia formation even though Rac is activated. Dominant-negative K44A dynamin-2 inhibited cell spreading and lamellipodia formation on fibronectin without blocking cell adhesion; dynamin-2 depletion by specific small interfering RNA inhibited lamellipodia in a similar manner. Dyn2(K44A) induced Rac mislocalization away from cell edges, into abnormal dorsal ruffles, and led to increased total Rac activity. Fluorescence resonance energy transfer imaging of Rac activity confirmed its predominant localization to aberrant dorsal ruffles in the presence of dominant-negative dyn2(K44A). Dyn2(K44A) induced the accumulation of tubulated structures bearing membrane-bound Rac-GFP. Constitutively active but not wild-type GFP-Rac was found on macropinosomes and Rac-dependent, platelet-derived growth factor-induced macropinocytosis was abolished by Dyn2(K44A) expression. These data suggest an indispensable role of dynamin in Rac trafficking to allow for lamellipodia formation and cell spreading.
Abbreviations used: FN, fibronectin; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; GST, glutathione S-transferase; HRP, horseradish peroxidase; PBD, p21-binding domain of PAK1; PDGF, platelet-derived growth factor; PRD, proline-, arginine-rich domain; siRNA, small interfering RNA; WT, wild type.
Online version of this article has video material for some figures. Online version is available at www.molbiolcell.org.
Present address University Eye Hospital, Division of Experimental Ophthalmology, Josef-Schneider Strasse 11, D-97080 Würzburg, Germany.
|| Present address Departments of Microbiology and Biomedical Engineering, Cardiovascular Research Center, University of Virginia, Charlottesville, VA 22908
¶ Corresponding author. E-mail: maschwartz{at}virginia.edu.
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