QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 26 February 2008
doi: 10.1242/jcs.020941

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Iwanicki, M. P. Articles by Parsons, J. T. Search for Related Content PubMed PubMed Citation Articles by Iwanicki, M. P. Articles by Parsons, J. T.

FAK, PDZ-RhoGEF and ROCKII cooperate to regulate adhesion movement and trailing-edge retraction in fibroblasts

¹ Department of Microbiology, University of Virginia Health System, Charlottesville, VA 22908, USA
² Department of Neurobiology and Anatomy, West Virginia University, Morgantown, WV 26506, USA
³ Departments of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA

View larger version (74K): [in this window] [in a new window]   Fig. 1. Serum starvation of NIH3T3 cells results in failure of trailing-edge retraction. (A) Length of the trailing edge and protrusion rates in NIH3T3 cells stably expressing βactin-mRFP cultured without serum or without serum but plus 2 µM LPA. For trailing-edge measurement, cells were filmed for 6 hours immediately after serum removal with images acquired at 5-minute intervals, using phase-contrast microscopy. Images acquired at the end of the sixth hour were quantified are shown. Data represent the analysis of 60 cells from two different experiments. For protrusion of the lamellipodia, cells were starved for 6 hours and at the end of sixth hour, images of βactin-mRFP in protruding lamellipodia were obtained for 20 minutes at 20-second intervals. The data represent analysis of six protruding lamellipodia per experimental condition. (B) Phase-contrast images and quantification of cell elongation of serum-starved cells, serum-starved cells treated with 2 µM LPA or 100 nM PDGF. Data represent the analysis of 50-72 cells per condition from two different experiments. (C) Kinetics of changes in cell elongation following LPA stimulation of serum-starved fibroblasts.

View larger version (30K): [in this window] [in a new window]   Fig. 2. LPA stimulation of adhesion movement. Rat2 or NIH3T3 cells stably expressing GFP-FAK or paxillin-GFP were plated in the presence of serum-containing (10%) medium on dishes coated with 1 µg/ml fibronectin. The following day, cells were placed in serum-free medium for 12 hours and subsequently stimulated with 2 µM LPA. (A) Analysis of adhesion movement in response to LPA. Serum starved NIH3T3 cells stably expressing paxillin-GFP were stimulated with 2 µM LPA and the movement of adhesions was analyzed using TIRF microscopy as described in Materials and Methods. Cells were filmed for 7 minutes before addition of LPA and 7 minutes after addition of LPA (arrows indicate representative adhesions). Adhesions present at time 0 were pseudo-colored green (left panels) and adhesions present at 7 minutes were colored red (middle panels). Movement of the individual adhesions was revealed using the merged images (right panels) and analyzed as described in Materials and Methods. (B) Quantification of adhesion movement by LPA in NIH3T3 cells expressing paxillin-GFP was determined as described in Materials and Methods. The experiment was performed three times, and five to seven cells were analyzed per experimental condition. (C) Western blot analysis of FAK activation by LPA in Rat2 cells stably expressing GFP-FAK. Cells were stimulated with LPA for 5 minutes and GFP-FAK phosphorylation was assessed using antibody against phosphorylated tyrosine (pTyr) as described in Materials and Methods. The increase in activation of MAP kinase was determined by immunoblotting using antibodies against phosphorylated and total ERK (pERK and ERK, respectively). (D) LPA activation of endogenous FAK in Rat2 cells. Cells were stimulated with LPA for 5 minutes and western blots were probed with antibody against phosphorylated Tyr397 (pY397) or anti-FAK antibody as described in Materials and Methods.

View larger version (63K): [in this window] [in a new window]   Fig. 3. Knockdown of FAK expression induces cell elongation. (A,B) Phase-contrast images, western blot analysis of FAK protein expression and quantification of cell elongation in control cells, FAK-siRNA treated Rat2 cells (A) or FAK-shRNA treated NIH3T3 cells (B). The extent of FAK knockdown was determined by western blotting and compared with expression of paxillin. (C) Western blot analysis of FAK expression in wild-type Rat2 cells, Rat2 cells expressing LacZeo and Rat2 cells expressing GFP-tagged chicken FAK. Endogenous FAK expression was substantially reduced in wild-type (wt), control-transfected cells (Lzeo) and in cells expressing GFP-tagged chicken FAK (GFP-FAK) following treatment with FAK siRNA (right three lanes) as compared with control siRNA-treated cells (left three lanes).The level of expression of GFP-tagged chicken FAK remained unchanged upon treatment with either control or FAK siRNA. Actin levels indicate the equal loading of sample in each lane. (D) Expression of GFP-FAK decreases cell elongation following FAK knockdown using siRNA. The images on the left illustrate the reduced immunostaining of FAK observed in cells treated with FAK siRNA. The images on the right illustrate the continued expression of GFP-tagged chicken FAK (GFP-FAK) in cells treated with control and FAK siRNA. Graphs panels A, B and D show cell-shape measurements that were assessed by determining the elongation factor for cells treated with either siRNAs or shRNAs.

View larger version (45K): [in this window] [in a new window]   Fig. 4. LPA-induced adhesion movement is FAK dependent. (A) Analysis of adhesion movement in response to 2 µM LPA. Control or FAK-siRNA-treated Rat2 cells stably expressing paxillin-GFP were plated on fibronectin-coated glass T dishes i medium containing 10% serum. The following day, cells were serum-starved for 12 hours and stimulated with 2 µM LPA. Adhesion movement was analyzed using TIRF microscopy as described in Fig. 3. Cells were filmed for 27 minutes before the addition of LPA and 27 minutes after the addition of LPA. (B) Quantification of dynamic adhesions was assessed as described in Materials and Methods (arrows indicate representative adhesions). The experiment was performed three times, six to eight cells were analyzed per experimental condition.

View larger version (26K): [in this window] [in a new window]   Fig. 5. FAK is required for LPA-mediated induction of trailing-edge retraction and the restoration of normal morphology. (A) Representative images of and -tubulin staining of control and FAK-siRNA-treated cells that were either serum starved, or serum starved and stimulated with 10% serum or 2 µM LPA. (B) The elongation factor for siRNA-treated cells was determined after treating cells that had been serum starved overnight with serum or with LPA for 7 hours. The experiment was repeated twice and 50-60 cells were analyzed per condition.

View larger version (28K): [in this window] [in a new window]   Fig. 6. Ectopic expression of PDZ-RhoGEF and RhoA-effector-domain mutant rescues elongation induced by knockdown of FAK expression. (A) NIH3T3 cells were transiently transfected with Myc-PDZ-RhoGEF, Myc-p115RhoGEF and Myc-PDZ-RhoGEF(1-585). Twenty-four hours after transfection, lysates were prepared and blotted with anti-Myc antibody. (B) Quantification of cell elongation. The shape of FAK-siRNA-treated cells that express different RhoGEF constructs was determined by measuring the elongation factor for individual cells. (C) FAK-shRNA-treated cells were transiently transfected with control non-functional PDZ-RhoGEF(561-585)-GFP, G14VRhoA/F39A-GFP or G14VRhoA/F39V-GFP. Twenty-four hours after transfection, lysates were prepared and blotted using anti-GFP antibody. The expression of GFP-tagged proteins was determined by immunoblotting individual lysates with anti-GFP and with anti-ERK antibodies to ensure equal loading of lysate samples. (D) Quantification of cell elongation in FAK-shRNA-treated cells expressing different RhoA-effector-domain mutants.

View larger version (46K): [in this window] [in a new window]   Fig. 7. GFP-FAK co-immunoprecipitates and localizes with co-expressed Myc-PDZ-RhoGEF and Myc-PDZ-RhoGEF(1-585). (A) HEK293T cells were transfected with with 0.5 µg GFP-FAK and Myc-PDZ-RhoGEF(1-585) or Myc-p115RhoGEF. To measure the amount of FAK in complex with the individual GEF proteins, immune complexes were isolated from lysates using anti-Myc antibody, and subjected to SDS-PAGE and western blotting with anti-FAK (top panel) or anti-GFP (second panel) antibodies. The expression of GEF proteins and GFP-FAK in lysates was determined by SDS-PAGE and immunoblotting lysates directly with anti-Myc or anti-GFP antibodies (bottom two panels). (B) Rat2 cells stably expressing GFP-FAK were transiently transfected with Myc-PDZ-RhoGEF, Myc-PDZ-RhoGEF(1-585) or Myc-p115RhoGEF, detached and seeded on fibronectin coated glass T dishes, fixed and Myc stained visualized using TIRF microscopy. Images show the colocalization of GFP-FAK and Myc-tagged PDZ-RhoGEF (top and middle panels), and the lack of colocalization of GFP-FAK and Myc-tagged p115RhoGEF. (C) Western blot analysis of endogenous PDZ-RhoGEF expression and phase-contrast images of control Rat2 cells and Rat2 cells treated with PDZ-RhoGEF siRNA. (D) Fluorescent images and cell elongation quantification of control Rat2 cells and Rat2 cells treated with PDZ-RhoGEF siRNA.

View larger version (61K): [in this window] [in a new window]   Fig. 8. PDZ-RhoGEF is required for LPA induction of adhesion movement. (A) Analysis of adhesion movement in response to LPA. Control Rat2 cells and Rat2 cells treated with PDZ-RhoGEF siRNA that stably expressed paxillin-GFP were plated on fibronectin-coated dishes in the presence of medium containing 10% serum. The following day, cells were serum starved for 12-16 hours and then stimulated with 2 µM LPA. Adhesion dynamic was analyzed using TIRF microscopy as described in Materials and Methods. Cells were filmed for 21 minutes before the addition of 2 µM LPA and 27 minutes after the addition of 2 µM LPA. (B) Quantification of dynamic adhesions (arrows indicate representative adhesions). The experiment was performed twice with five to seven cells analyzed per condition.

View larger version (64K): [in this window] [in a new window]   Fig. 9. LPA induced adhesion movement requires ROCKI and ROCKII. (A) Western blot analysis of ROCKI and ROCKII protein expression in control NIH3T3 cells and NIH3T3 cells treated with shRNA targeting FAK, ROCKI or ROCKII (top panel). Micrographs show phase-contrast images of control NIH3T3 cells and NIH3T3 cells treated with shRNA targeting ROCKI or ROCKII. (B) Control NIH3T3 cells and NIH3T3 cells treated with shRNA targeting ROCKI or ROCKII were serum starved for 12 hours. Following starvation, cells were stimulated with 2 µM LPA and adhesion movement was visualized by TIRF microscopy. (C) Quantification of adhesion movement (as described in Materials and Methods) in unstimulated serum-starved cells and in serum-starved cells stimulated with LPA (arrows indicate representative mobile adhesions). The experiment was performed twice with six to eight cells analyzed per experimental condition. (D) Quantification of cellular elongation in LPA-stimulated control cells and ROCKII-shRNA-treated cells. The experiment was performed twice.