BMM (top panel) or RAW/LR5 cells (bottom panel) were treated or not with 20 ng/ml CSF-1 for 5 minutes and then fixed and stained for F-actin using Alexa 568-phalloidin and for IRSp53 using a polyclonal goat antibody followed by Alexa 488-anti-goat IgG. Scale bar = 10 µm.

(A) The level of IRSp53 and WAVE2 present in IRSp53 shRNA-treated (IRSp53 sh) RAW/LR5 cells was analyzed by western blotting with the indicated antibodies and compared to mock shRNA treated cells. A representative blot and quantification of IRSp53/β-actin signal intensity ratios are shown, n = 3, *: p < 0.05 compared to mock shRNA-treated cells. (B) The level of IRSp53 present in IRSp53 shRNA-treated (IRSp53 sh) RAW/LR5 cells was analyzed by immunofluorescence with the indicated antibodies and compared to mock shRNA treated cells. Representative images and quantification of IRSp53 signal intensity are shown, n = 3, *: p < 0.05 compared to mock shRNA-treated cells. (C) Mock or IRSp53 shRNA-treated RAW/LR5 cells were treated with or without CSF-1 for 5 minutes and F-actin rich protrusions were visualized by Alexa 568-phalloidin staining. Scale bar = 10 µm. (D) The number of CSF-1-elicited protrusions in mock (white bar) or IRSp53 shRNA-treated RAW/LR5 cells from C was quantified and expressed as percent of the CSF-1 stimulation observed in mock shRNA-treated cells, n = 3, *: p < 0.05 compared to mock shRNA-treated cells. The dotted line represents basal ruffling. (E)Mock (white bar) or IRSp53 shRNA-treated (grey bar) RAW/LR5 cells were fixed after treatment with or without CSF-1 and total F-actin content, normalized to the cell number, was quantitatively measured as described in Materials and Methods and compared to unstimulated control cells, n = 3, *: p < 0.05 compared to mock shRNA-treated cells. (F) Chemotaxis and chemokinesis in response to CSF-1 in Mock (white bars) or IRSp53 shRNA-treated (grey bars) RAW/LR5 cells was tested using a transmigration chamber assay as described in Materials and Methods. CSF-1-stimulated migration of each cell population was compared to the corresponding unstimulated condition and expressed as a fold induction. n = 3, *: p < 0.05 compared to mock shRNA-treated cells.

(A)Representative images of F-actin stained mock or IRSp53 shRNA-treated non transfected (top panel) or expressing either Myc/Rac1Q61L (middle panel) or Myc/Cdc42V12 (bottom panel) RAW/LR5 cells are shown where cells were stained for Myc to identify expressing cells. Scale bar = 10 µm. The ability of Mock (white bars) or IRSp53 shRNA-treated (grey bars) cells to either exhibit ruffles following Rac1Q61L expression (B)or to exhibit filopods following Cdc42V12 expression (C)was quantified and expressed as percent of total cells counted, n = 3, *: p < 0.05 compared to mock shRNA-treated cells. Scale bar = 10 µm.

(A)Lysates from non-transfected or GFP-tagged IRSp53 and Myc-tagged Rac1 or Rac1N17 or Rac1Q61L or Cdc42V12 co-expressing RAW/LR5 cells and Cos7 cells were immunoprecipitated with antibodies either against GFP or against Myc (IP) and subjected to western blotting using the indicated antibodies. Representative example of three independent experiments is shown.(B) Lysates from Mock or IRSp53 shRNA-treated RAW/LR5 cells expressing Myc-tagged Rac1Q61L were immunoprecipated with antibodies against Myc (IP) and were then subjected to western blotting using the indicated antibodies. (C) Quantification of WAVE2 or Abi1 co-immunprecipitated by Myc (from B) is shown, n = 3,*: p < 0.05 compared to mock shRNA-treated cells expressing Rac1Q61L (D)Lysates from non transfected or Myc-tagged Rac1Q61L and HA-tagged IRSp53ΔSH3 co-expressing RAW/LR5 cells were incubated with antibodies against HA for immunoprecipitation (IP-HA), followed by sequential immunoprecipitation of Rac1Q61L using antibodies against Myc (IP-Myc). Immunoprecipitates were then subjected to western blotting using the indicated antibodies. Mock or IRSp53 shRNA-treated (E)or WAVE2 shRNA-treated (F) RAW/LR5 cells either transiently transfected with the indicated constructs or not, were stimulated with CSF-1 and their ability to form F-actin rich protrusions in response to CSF-1 was analyzed as in 2D and expressed as percent of the CSF-1 stimulation observed in non transfected mock shRNA-treated cells, n = 3, *: p < 0.05 compared to non transfected mock shRNA-treated cells (represented by the dotted line).

(A)Confocal images of RAW/LR5 cells expressing either FLAG-tagged WAVE2 (left) or WAVE2CAAX (right) are shown with single XY, XZ and YZ cross sections through different areas of the cell as indicated by the cross-hair lines. Scale bar = 10 µm (B)RAW/LR5 cells expressing either FLAG-tagged WAVE2 or WAVE2CAAX were fixed and stained for FLAG and F-actin and the total F-actin content was quantified as described in Materials and Methods and compared to non transfected cells, n=3. (C)FLAG-tagged WAVE2CAAX expressing RAW/LR5 cells were treated or not with CSF-1 for 5 minutes. The number of CSF-1-elicited protrusions was quantified as in 2D and expressed as percent of the CSF-1 stimulation observed in non transfected cells on the same coverslip, n = 3. (D)RAW/LR5 (white bars) and Cos-7 (grey bars) cells were either transfected with Myc-tagged Rac1Q61L (plain) or RacQ61L and FLAG-tagged WAVE2 CAAX (striped) constructs. Co-expressing cells were identified by the Myc and FLAG staining and the ability of cells to either exhibit ruffles was quantified and expressed as percent of total cells counted, n = 3. (E)Mock or WAVE2 shRNA-treated RAW/LR5 cells either transiently transfected with the indicated constructs or not, were stimulated with CSF-1 and their ability to form F-actin rich protrusions in response to CSF-1 was analyzed as in 2D and expressed as percent of the CSF-1 stimulation observed in non transfected mock shRNA-treated cells, n = 3, *: p < 0.05 compared to non transfected mock shRNA-treated cells (represented by the dotted line).

(A)Based on several reports a model for the regulation of WAVE2 has been proposed where the stable WAVE2/Abi1 complex is constitutively active in the cytoplasm in vivo. Upon receptor tyrosine kinase (RTK) stimulation by growth factor (GF), activated Rac1 binds to WAVE2, mediated by PIR121 in the Abi1 complex, and induces its translocation to the leading edge where it stimulates site restricted actin polymerization necessary for cell protrusion and motility. (B) Alternately, the stable WAVE2/Abi1 complex is inactive in the cytoplasm in vivo. Upon CSF-1 receptor (CSF-1 R) stimulation by CSF-1, activated Rac1 binds to WAVE2, mediated by IRSp53, and induces its activation and translocation to the leading edge where it stimulates a site restricted actin polymerization required for cell protrusion and motility. Abbreviations: WHD, WAVE homology domain; B, basic region; Pro-rich, proline rich region; V, verprolin homology domain; C, cofilin homology domain; A, acidic region.