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First published online 17 February 2009
doi: 10.1242/jcs.032094

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Memo is a cofilin-interacting protein that influences PLC1 and cofilin activities, and is essential for maintaining directionality during ErbB2-induced tumor-cell migration

¹ Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland
² Terrence Donnelly Centre for Cellular and Biomolecular Research (CCBR), Department of Biochemistry and Department of Molecular Genetics, University of Toronto, Toronto, M5S 3E1, Ontario, Canada

View larger version (33K): [in this window] [in a new window]   Fig. 1. PLC1 and Memo are required for ErbB2-mediated cell motility. (A) (Left panel) SKBr3 cells were transfected with lacZ, Memo and/or PLC1 siRNAs. Control responses in the absence of ligand, as well as chemotactic responses towards 1nM HRG and chemokinetic responses in the presence of 1 nM HRG, were determined in Transwell assays. (B) (Left panel) Immunofluorescence microscopy of HRG-stimulated T47D cells treated with lacZ, Memo or/and PLC1 siRNAs and labeled with Alexa-Fluor-488-phalloidin (green) or an anti--tubulin (red) antibody. Yellow arrows indicate the dense array of actin stress fibers. (A and B, right panels) Extracts were prepared and analyzed for Memo and/or PLC1 KD; -tubulin was used as a control. The data are representative of several independent experiments. Scale bar, 10 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 2. Effect of Memo and PLC1 KD on HRG-induced chemotaxis in Dunn chambers. T47D cells were transiently transfected with lacZ, Memo or PLC1 siRNAs and 3 days later were analyzed for their ability to migrate in a gradient of HRG. (A) Track displacement was followed during a period of 12 hours. The gradient follows the path from the bottom left to the top right. Gray arrows indicate the direction of individual cells. Cell displacement over the time course is indicated with the colored line (zero, intermediate and final time points are in blue, red and yellow, respectively). KD of Memo or PLC1 was confirmed by western blot analysis, -tubulin was loaded as a control. (B) (Left panel) The net translocation distance is shown as the mean ± s.e.m. of the displacement of 61 cells. (Middle panel) The migration speed is shown as the mean ± s.e.m. of the paths of 61 cells. (Right panel) The directional persistency index of control lacZ, Memo or PLC1 KD cells is shown. (C) Decagonal histograms indicating the distribution of cells at their final location. The source of HRG is at the top. The data are representative of several independent experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Memo complexes with PLC1 and ErbB2 and regulates PLC1 phosphorylation in tumor cells. (A) (Left panel) T47D cells were stimulated or not with 10 nM HRG for 5 minutes and cell extracts were subjected to immunoprecipitation using a Memo mAb. IPs were probed for PLC1 and Memo; a mouse IgG2a mAb was used as a control. (Right panel) T47D cells were stimulated or not with 10 nM HRG for 5 minutes. Cell extracts were subjected to immunoprecipitation using a PLC1 antibody and IPs were probed for PLC1 and Memo. Whole-cell lysates (W) were loaded as controls. (B) (Left panel) T47D cells were stimulated or not with 10 nM HRG for 5 minutes. Cell extracts were subjected to immunoprecipitation using a PLC1 mAb; IPs were probed for Tyr-P and for PLC1. (Middle and right panels) T47D cells were transiently transfected with lacZ or Memo siRNA, and stimulated or not for 5 minutes with 10 nM HRG. Cell extracts were subjected to immunoprecipitation using a PLC1 mAb (middle panel). PLC1 phosphorylation was analyzed by western blotting using a Tyr-P mAb. Blots were reprobed for PLC1. (Right panel) Memo and PLC1 levels in transfected cells were monitored in whole-cell lysates (W) using the respective antibodies.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Memo interacts with cofilin. (A) YTH analysis. The yeast reporter strain L40 was co-transformed with a plasmid encoding the LexA binding domain fused to Memo (pLexAKan-Memo) and a plasmid encoding GAL4 activation domain fused to the cofilin cDNA (pACT-cofilin) (lower lane). The negative controls are: transformation with pLexAKan-Memo and the empty prey plasmid (2nd row) or pACT-BLM encoding a nuclear helicase (3rd row). β-galactosidase (β-Gal+) strain was used as a positive control (top row). (B) Pull-down assay of GST-Memo–cofilin. Immobilized GST-Memo on glutathione-Sepharose beads was incubated with (lane 4) or without (lane 5) 0.24 nmoles of recombinant human cofilin. A five- or tenfold molar excess of soluble Memo (lanes 7 and 8, respectively) or a fivefold molar excess of BSA (lane 9) was added to cofilin 1 hour prior to incubation with the GST-Memo immobilized beads. As a control, cofilin was incubated with the glutathione-Sepharose beads (lane 6). After incubation, the beads were pelleted, washed, and the bound proteins were eluted and loaded on an SDS-gel. The gel was stained with 0.1% CBB. GST-Memo immobilized beads, glutathione-Sepharose beads and cofilin were loaded on the same gel (lanes 1, 2 and 3, respectively). (C) T47D (upper and lower panels) and MDA-MB435 (middle panel) cells were stimulated or not with 10 nM HRG for 5 minutes. Memo was immunoprecipitated using a mouse mAb; a mouse mAb IgG2a was used as a negative control. Co-immunoprecipitating proteins were analyzed by western blotting and probed with ErbB2, Memo, cofilin or cofilin-P polyclonal antibodies. Whole-cell lysates (W) were loaded as controls. (D) T47D cells were treated with 10 nM HRG for the indicated times. Cell extracts were subjected to immunoprecipitation using a Memo mAb, and IPs were probed for ErbB2, PLC1, Memo and cofilin. Whole-cell lysates were loaded as controls.

View larger version (28K): [in this window] [in a new window]   Fig. 5. PLC1, Memo and cofilin are required for ErbB2-mediated directional migration in Dunn chambers. T47D cells were transiently transfected with either lacZ or cofilin siRNAs, or simultaneously transfected with Memo and PLC1 siRNAs. Cell migration was analyzed in a gradient of HRG in Dunn chambers for 4.5 hours. Net translocation distance and migration speed were calculated for 78 cells. Cofilin, Memo and PLC1 levels in KD cells were checked by western blotting with the respective antibodies. -Tubulin levels were monitored as a control. The data are representative of several independent experiments.

View larger version (47K): [in this window] [in a new window]   Fig. 6. HRG-induced recruitment of cofilin to the lamellipodia is dependent on Memo and PLC1. (A) GFP-cofilin-transfected and (B) GFP-Memo-transfected SKBr3 were stimulated or not with 1 nM HRG for 20 minutes. Cells were stained with a TRITC-labeled phalloidin antibody. (C) SKBr3 cells were simultaneously transfected with GFP-cofilin and lacZ, Memo or PLC1 siRNAs and HRG-dependent recruitment of GFP-cofilin to the lamellipodia was visualized. The actin cytoskeleton was examined with a TRITC-labeled phalloidin antibody. (D) SKBr3 cells were simultaneously transfected with GFP-Memo and siRNAs for lacZ, cofilin or PLC1 and HRG-dependent recruitment of GFP-Memo to the lamellipodia was visualized. The actin cytoskeleton was examined with a TRITC-labeled phalloidin antibody. Shown are representative images from two independent experiments visualizing 30-40 GFP-stained cells. Merged images are shown in the bottom panels. Scale bar, 10 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 7. F-actin binding experiments. For each reaction, samples of the supernatant (S) and pellet (P) fractions were collected and separated on a 10% SDS-gel. The gel was stained with 0.1% CBB and quantified for actin, Memo and cofilin levels. The molar ratios of cofilin or Memo to actin were: cofilin:actin, 0.25:1, memo:actin, 0.05:1. (Upper panel; left) F-actin binding assay. Reactions: 1, actin; 2, -actinin; 3, -actinin and actin; 4, BSA and actin; 5, cofilin; 6, actin and cofilin (A+C); 7, Memo (M); 8, Memo and cofilin; 9, actin and Memo (A+M); 10, actin, Memo, and cofilin (A+M+C). (Upper panel; right) Fold increase of actin detected in the supernatant compared with reaction 1. (Lower panel) Quantification of cofilin (left) and Memo (right) levels in the supernatant and pellet fractions.

View larger version (24K): [in this window] [in a new window]   Fig. 8. Effect of Memo on cofilin-induced actin depolymerization and severing. (Upper panel) For each reaction, supernatant (S) and pellet (P) fractions were collected, separated on an SDS-gel and quantified after staining of the gel with 0.1% CBB. Molar ratios of cofilin or Memo to actin were the following: cofilin:actin, 1:1, memo:actin, 0.125:1, or 0.25:1. Reactions: 1, actin; 2, actin and cofilin; 3, actin and Memo 4.8 µg; 4, actin and Memo 9.6 µg; 5 to 6, actin and cofilin plus increasing Memo amounts (4.8 and 9.6 µg). The picture is representative of several independent experiments. (Lower left panel) Quantification of the fold increase of actin in the supernatant fractions compared with reaction 2. (Lower right panel) Quantification of the fold increase of cofilin in the supernatant fractions compared with reaction 2. Both quantifications are shown as the mean ± s.e.m. of three independent experiments. P-values are indicated.

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