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First published online 24 May 2005
doi: 10.1242/jcs.02380

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Focal adhesion kinase is required for the spatial organization of the leading edge in migrating cells

¹ Department of Microbiology and Cancer Center, University of Virginia Health System, Charlottesville, VA 22908, USA
² Howard Hughes Medical Institute and Department of Physiology, University of California, San Francisco, CA 94143, USA

View larger version (58K): [in a new window]   Fig. 1. Treatment of REF52 cells with siRNAs for FAK results in loss of FAK expression and inhibition of focal adhesion protein phosphorylation. (A) REF52 cells were untreated, or treated with either luciferase or FAK siRNA, and equal amounts of lysates were analyzed by western blot 72 hours later for FAK expression (a). The blot was stripped and reprobed with an anti-phosphotyrosine antibody (P-Tyr, b), anti-phospho-FAK 397 (c), anti-phospho paxillin Y31 (d) and anti-phospho-paxillin Y118 (e). The blot was also stripped and re-probed for total paxillin (f) and p130Cas levels (g) as loading controls. Cells were either kept in suspension (S) or allowed to adhere to fibronectin (FN) for the indicated times. (B) Cells treated with either the luciferase siRNA (a,c) or FAK siRNA (b,d) were allowed to adhere to fibronectin-coated coverslips and immunofluorescently stained for either FAK (a,b) or actin (c,d). The arrows (a) denote focal adhesions.

View larger version (91K): [in a new window]   Fig. 2. FAK siRNA knockdown results in abnormal cell spreading on fibronectin. REF52 cells were treated with either luciferase siRNA (A-C, G-I) or FAK siRNA (D-F, J-L). The cells were plated on fibronectin for the indicated times either in the absence (A-F) or presence (G-L) of 10% serum and stained for actin (green) and paxillin (red). Arrows in C and I indicate the smooth membrane of the control cells; arrowheads in L indicate the projections in the knockdown cells.

View larger version (95K): [in a new window]   Fig. 3. Wild-type FAK, but not FAK Y397F, rescues the phenotype resulting from FAK knockdown. Cells treated with siRNAs for FAK were transiently transfected with either wtFAK-GFP (A-D) or FAK Y397F-GFP (E-H) and plated on fibronectin for 1 hour. The images show GFP-FAK (A-B, E-F) and the corresponding phase photographs (C-D, G-H). Arrows indicate cells that are expressing the GFP-FAK constructs.

View larger version (42K): [in a new window]   Fig. 4. Aberrant lamellipodia formation in cells treated with FAK siRNAs. Cells treated with either luciferase siRNA (A) or FAK siRNA (B) were allowed to spread on fibronectin and analyzed by time-lapse microscopy. Arrows in A indicate ruffling edge of the control cells; arrowheads in B indicate the formation of a projection in the FAK knockdown cells. (C) FAK knockdown cells were allowed to spread on fibronectin for 1 hour, then fixed and stained for paxillin. The white outline in the enlarged inset represents the plasma membrane.

View larger version (93K): [in a new window]   Fig. 5. The projections in the cells treated with FAK siRNAs are rich in cortactin and are dependent on functional Rac. Cells treated with either luciferase siRNA (A) or FAK siRNA (B-F) were allowed to spread on fibronectin for 1 hour. (A) Control cells stained for cortactin. (B) FAK knockdown cells stained for cortactin. Arrowheads indicate cortactin staining along the cell periphery in A and in the multiple cellular extensions in B. (C) Cortactin staining in FAK knockdown cells following re-expression of GFP-wtFAK. Arrow indicates cell transfected with GFP-wtFAK. D is the corresponding GFP image for C. (E-F) FAK knockdown cells expressing GFP-dnRac. E shows the GFP image and F shows the corresponding phase image. Arrows point to cells expressing GFP-dnRac.

View larger version (102K): [in a new window]   Fig. 6. Treatment of cells with FAK siRNAs results in a loss of directional movement. REF52 cells were treated with either luciferase siRNA (A) or FAK siRNA (B). A confluent culture of REF52 cells was wounded using a pipette tip, and cell migration into the wound was monitored by time-lapse microscopy. Images correspond to photographs taken at the indicated times following the scraping of the monolayer. In A, the cells develop broad lamellipodia (arrows) as they move into the wound. In B, cell `a' has migrated into the wound yet did not release its trailing edge, and cell `b' eventually has become elongated as it develops two leading edges, one moving toward the wound and the other away from it. Both cells lack broad lamellipodia. See Movies 1 and 2 in supplementary material.

View larger version (94K): [in a new window]   Fig. 7. Treatment of Rat-2 cells with FAK siRNAs inhibits spontaneous polarization. Rat-2 cells were either transfected with luciferase siRNA (A) or FAK siRNA (B) and allowed to spread on fibronectin for 10 minutes before video time-lapse microscopy. Images were taken at the indicated timepoints after the initiation of the recording. The arrows in A indicate the broad lamellipodia at the leading edge of the migrating cells; the arrowheads in B indicate the multiple extensions that form in the FAK knockdown cells. See Movies 3 and 4 in supplementary material.

View larger version (48K): [in a new window]   Fig. 8. Golgi re-orientation is impaired in FAK knockdown cells. Rat-2 cells treated with either luciferase (A) or FAK (B) siRNA were subjected to a wound-healing assay for 4 hours, then stained for Golgi (red) and nuclei (blue), and analyzed for Golgi reorientation as described in Materials and Methods. The white line represents the location of the wound. Arrows in B indicate the disorganized Golgi in the FAK knockdown cells. (C) The percentages of control (white bars) and FAK knockdown (black bars) cells with Golgi orientated toward the wound were calculated at 2 and 4 hours following wounding of the monolayer. Error bars indicate standard deviation. *P<0.05 compared with the control bars.

View larger version (116K): [in a new window]   Fig. 9. Cre-mediated deletion of FAK in primary fibroblasts results in impaired leading edge formation. (A) PCR analysis of wild-type (lanes 1-2), homozygous floxed (lanes 3-4) or heterozygous floxed (lanes 5-6) fibroblasts. Lanes 1, 3 and 5 show the PCR products from DNA of cells that were not exposed to the Cre-containing adenovirus; lanes 2, 4 and 6 show cells the products from cells infected with adeno-Cre. Arrow points to the band corresponding to the expected PCR product from the intact FAK/Lox sequence; arrowhead indicates the PCR product from the recombined sequence following Cre-mediated excision. (B) Western blot of cellular lysates from wild-type, floxed and heterogeneous WT/Flox fibroblasts. Arrow indicates the band corresponding to FAK. The blot was stripped and probed for ERK as a loading control. Monolayers of either wild-type (C) or Floxed (D) fibroblasts were wounded and analyzed by video time-lapse microscopy. The white outlines denote the plasma membranes of the cells that started from the edge of the wound at the bottom of the photograph. See Movies 5 and 6 in supplementary material.

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