Volume 16, Issue 14, Pages 1375-1385 (July 2006) CdGAP Associates with Actopaxin to Regulate Integrin-Dependent Changes in Cell Morphology and Motility  David P. LaLonde, Markus Grubinger, Nathalie Lamarche-Vane, Christopher E. Turner  Current Biology  Volume 16, Issue 14, Pages 1375-1385 (July 2006) DOI: 10.1016/j.cub.2006.05.057 Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 1 CdGAP Localizes to Focal Adhesions (A) U2OS osteosarcoma cells were cotransfected with GFP-CdGAP and RFP-actin. GFP-CdGAP was imaged in live cells by using Total Internal Reflection Fluorescence (TIRF) microscopy, whereas the RFP-actin image was obtained by using epifluorescence. CdGAP localizes to the ends of F-actin-containing stress fibers. Scale bar represents 10 μm. (B) Further examples of cells imaged as in (A). Merged images represent GFP-CdGAP (green) and RFP-actin (red) in U2OS cells. (C) U2OS cells were cotransfected with GFP-CdGAP and DsRed-paxillin and processed for microscopy as above. CdGAP is shown to localize to focal adhesions. Current Biology 2006 16, 1375-1385DOI: (10.1016/j.cub.2006.05.057) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 2 CdGAP's GAP Activity Is Regulated by Integrin Ligation (A) U2OS cells transfected with GFP-CdGAP were lysed either in suspension or at 15, 30, 60, or 150 min after adhesion to 10 μg/ml collagen. GFP immunoprecipitations were then performed, and the immune complexes were subjected to in vitro GAP assays with (γ-32P) GTP-loaded Rac as the substrate, with less bound GTP indicating higher GAP activity. The inset blot confirms that equal amounts of GFP-CdGAP were precipitated for each time point. The control reaction was performed from cells transfected with GFP and indicates the baseline hydrolysis of GTP. ∗ indicates a significant statistical activation of CdGAP at 60 and 150 min as compared to the activity levels found in suspended cells, p < 0.05. Error bars represent the standard deviation. (B) U2OS cells expressing the indicated constructs were lysed, followed by GFP immunoprecipitations and in vitro GAP assays. CdGAP-R56AN169V has a significant reduction of GAP activity as compared to wild-type CdGAP. Error bars represent standard deviation from four separate experiments. ∗ indicates significantly more hydrolysis than GFP control, p < 0.001. Expression levels of constructs are illustrated by western blotting for GFP. Current Biology 2006 16, 1375-1385DOI: (10.1016/j.cub.2006.05.057) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 3 CdGAP Activity Inhibits Cell Spreading (A) U2OS cells transfected with GFP, GFP-CdGAP, or GFP-CdGAP R56AN169V were spread on 10 μg/ml collagen-coated coverslips, fixed at 120 min, and stained for F actin. CdGAP-overexpressing cells display decreased spreading, whereas CdGAP R56AN169V-expressing cells are well spread and often display multiple protrusions. Scale bar represents 10 μm. (B) U2OS cells expressing the indicated constructs were spread on 10 μg/ml collagen-coated cover slips for 15, 30, 60, or 120 min and then processed for area quantitation. CdGAP overexpression inhibits spreading at all time points tested, whereas the GAP-deficient CdGAP R56AN169V construct significantly facilitates spreading at 15 and 30 min after adhesion, p < 0.01, as indicated by asterisks. Error bars represent the standard error of the mean (SEM). (C) An examination of the number of lamellipodia from cells respread as in (A) shows that CdGAP-overexpressing cells rarely develop a single dominant lamellipodia. CdGAP R56AN169V-expressing cells often have multiple lamellipodia. Error bars represent the standard deviation. (D) PBD assays were performed at 30 min after adhesion to determine the active Rac levels of U2OS cells transfected with the indicated constructs. CdGAP overexpression inhibits Rac activity, whereas this effect is lost with the GAP-deficient variant, CdGAP R56AN169V. Current Biology 2006 16, 1375-1385DOI: (10.1016/j.cub.2006.05.057) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 4 CdGAP Depletion Increases Cell Spreading (A) Endogenous CdGAP levels were depleted in HeLa cells by using two specific oligonucleotides (Oligo #1 and #2) or a nonspecific oligonucleotide as a control. Equivalent total protein is demonstrated by blotting for ILK. (B) HeLa cells that had received either control oligonucleotide or Oligo #2 were suspended and spread on fibronectin for 30 min and then fixed and stained for F actin. Cells depleted of CdGAP spread more efficiently. Scale bar represents 10 μm. (C) HeLa cells were spread on either 10 μg/ml collagen or 10 μg/ml fibronectin and then fixed at 30 min and stained for F actin. Cells depleted of CdGAP spread more efficiently on both matrices at 30 min. ∗ indicates a significant increase from control cells, p < 0.05. Error bars represent the SEM. (D) Control and CdGAP RNAi HeLa cells were evaluated for Rac activity at 30 min after adhesion on fibronectin by using PBD assays. Current Biology 2006 16, 1375-1385DOI: (10.1016/j.cub.2006.05.057) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 5 CdGAP Associates with the Focal-Adhesion Protein Actopaxin (A) U2OS cells transfected with Myc-CdGAP were subjected to binding assays with the indicated GST-actopaxin fusion proteins. Samples were analyzed via western blotting and probed for Myc and α-actinin, as a negative control. Myc-CdGAP was only precipitated by full-length or amino-terminal pieces of actopaxin. (B) GST-actopaxin binding assays were performed with cell lysates from U2OS cells transfected with the indicated CdGAP constructs. Actopaxin precipitated full-length CdGAP (aa 1–820), the full-length GAP-deficient mutant (aa 1–820 R56AN169V), an aa 182–820, or an aa 1–515 construct. Conversely, GST-actopaxin does not bind CdGAP aa 1–181, aa 182–515, or aa 516–820 constructs. (C) CdGAP and ILK were synthesized as 35S-labeled proteins by using an in vitro transcription/translation rabbit reticulocyte system and used in binding assays with GST, GST-actopaxin 1–372, or GST-actopaxin 1–95. Results demonstrate that the association between actopaxin and CdGAP is direct and occurs within the amino-terminal 95 amino acids of actopaxin. ILK was used as a specificity control because it binds to the actopaxin carboxyl terminus. (D) Endogenous actopaxin was precipitated from asynchronously growing U2OS cells. The lysate lane contained 20 μg protein, and immunoprecipitations were performed from 250 μg each. Endogenous CdGAP coprecipitated with actopaxin, and α-actinin served as a negative control. The control immunoprecipitation was performed with nonspecific Rabbit IgG. Approximately 10% of available actopaxin was precipitated, and 1% of endogenous CdGAP was coprecipitated. Current Biology 2006 16, 1375-1385DOI: (10.1016/j.cub.2006.05.057) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 6 The Actopaxin-CdGAP Interaction Is Required for Regulation of Normal Cell Spreading (A) U2OS cells transfected with Myc-CdGAP were lysed and subjected to binding assays with GST, GST-actopaxin, or GST-actopaxin Δ21–25. GST-actopaxin lacking amino acids 21–25 fails to associate with CdGAP but retains normal binding to ILK, and α-actinin served as a negative control. (B) U2OS cells were cotransfected with Myc-CdGAP and either Xpress-actopaxin or Xpress-actopaxin Δ21–25 followed by Omni-probe immunoprecipitations of the Xpress-tagged proteins. Xpress-actopaxin Δ21–25 was unable to bind CdGAP, but retains its association with ILK. (C) U2OS cells transfected with the indicated constructs were processed to evaluate their effects on cell spreading. Expression of actopaxin Δ21–25 significantly increases spreading at 15 and 30 min after adhesion, p < 0.05, as indicated by asterisks. Error bars represent the SEM. (D) Transfected U2OS cells were spread for 30 min on 10 μg/ml collagen, followed by area quantification. The coexpression of actopaxin Δ21–25 with CdGAP significantly reverses the inhibited spreading seen in cells overexpressing CdGAP alone, whereas the Xpress actopaxin S4/8D construct does not. All conditions are significantly different than GFP control cells, p < 0.05. Error bars represent the SEM. Current Biology 2006 16, 1375-1385DOI: (10.1016/j.cub.2006.05.057) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 7 Actopaxin Facilitates CdGAP Localization to Adhesions and Its Effects on Motility (A) U2OS cells were cotransfected with Myc-CdGAP and empty vector (EV), Xpress-actopaxin wild-type (WT), or Xpress-actopaxin Δ21–25. Cytoskeletal fractionation was performed from cells either growing asynchronously or spread on collagen for 1 hr. The levels of CdGAP are shown to increase in the cytoskeletal fraction during spreading. However, this increase is lost in cells expressing the actopaxin Δ21–25 mutant unable to bind CdGAP. (B) U2OS cells were cotransfected with GFP-CdGAP, DsRed-paxillin, and either wild-type actopaxin or actopaxin Δ21–25 and then spread on collagen for 1 hr. CdGAP localization to focal adhesions was then quantified as detailed in Experimental Procedures. The images shown are representative of CdGAP that is either localized or not localized to focal adhesions. The numbers represent percentage of cells exhibiting these localizations under each condition. (C) CdGAP overexpression inhibits random motility in transiently transfected U2OS cells. Expression of actopaxin Δ21–25 reverses the decreased motility observed in cells overexpressing CdGAP. Conversely, the actopaxin S4/8D construct causes increased random motility, but does not increase motility in CdGAP overexpressors. ∗ indicates a significant difference from GFP control cells, p < 0.01. ∗∗ indicates that Xpress-actopaxin Δ21–25 significantly rescues CdGAP motility defect, while still being significantly below the GFP control cells, p < 0.01. Error bars represent the standard deviation. Current Biology 2006 16, 1375-1385DOI: (10.1016/j.cub.2006.05.057) Copyright © 2006 Elsevier Ltd Terms and Conditions