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Vol. 20, Issue 21, 4509-4523, November 1, 2009

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Unbalancing the Phosphatidylinositol-4,5-bisphosphate–Cofilin Interaction Impairs Cell Steering

Shirley Leyman^*,,, Mazen Sidani^,, Laila Ritsma^||, Davy Waterschoot^*,, Robert Eddy, Daisy Dewitte^*,, Olivier Debeir^¶, Christine Decaestecker^¶,#, Joël Vandekerckhove^*,, Jacco van Rheenen^,||,@, Christophe Ampe^*,, John Condeelis^,@, and Marleen Van Troys^*,

*Department of Medical Protein Research, VIB, B-9000 Ghent, Belgium; Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, B-9000 Ghent, Belgium; Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461; ^@Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461; ^||Hubrecht Institute-KNAW and University Medical Center Utrecht, 3584 CT Utrecht, The Netherlands; ^¶Laboratory of Image Synthesis and Analysis (LISA), Faculty of Applied Sciences, Université Libre de Bruxelles, 1050 Brussels, Belgium; and ^#Laboratory of Toxicology, Institute of Pharmacy, Université Libre de Bruxelles, 1050 Brussels, Belgium

Submitted February 11, 2009; Revised August 27, 2009; Accepted August 31, 2009
Monitoring Editor: Carole Parent

Cofilin is a key player in actin dynamics during cell migration. Its activity is regulated by (de)phosphorylation, pH, and binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]. Here, we here use a human cofilin-1 (D122K) mutant with increased binding affinity for PI(4,5)P₂ and slower release from the plasma membrane to study the role of the PI(4,5)P₂–cofilin interaction in migrating cells. In fibroblasts in a background of endogenous cofilin, D122K cofilin expression negatively affects cell turning frequency. In carcinoma cells with down-regulated endogenous cofilin, D122K cofilin neither rescues the drastic morphological defects nor restores the effects in cell turning capacity, unlike what has been reported for wild-type cofilin. In cofilin knockdown cells, D122K cofilin expression promotes outgrowth of an existing lamellipod in response to epidermal growth factor (EGF) but does not result in initiation of new lamellipodia. This indicates that, next to phospho- and pH regulation, the normal release kinetics of cofilin from PI(4,5)P₂ is crucial as a local activation switch for lamellipodia initiation and as a signal for migrating cells to change direction in response to external stimuli. Our results demonstrate that the PI(4,5)P₂ regulatory mechanism, that is governed by EGF-dependent phospholipase C activation, is a determinant for the spatial and temporal control of cofilin activation required for lamellipodia initiation.

These authors contributed equally to this work.

Address correspondence to: Marleen Van Troys (leen.vantroys{at}ugent.be).

Abbreviations used: ADF, actin depolymerizing factor; EGF, epidermal growth factor; FLIP, fluorescence loss in photobleaching; FRET, fluorescence resonance energy transfer; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KD, knockdown; MDO, maximum distance from origin; M-KD, MTLn3 cells with cofilin knockdown and expressing D122K cofilin; PI(4,5)P₂, phosphatidylinositol-4,5-bisphosphate; PLC, phospholipase C; PM, plasma membrane; W-KD, MTLn3 cells with cofilin knockdown expressing wild-type cofilin; WT, wild type.