WAVE2 is enriched in F-actin rich membrane protrusions elicited by CSF-1 in macrophages. BMM (A) or RAW/LR5 cells (B) were left untreated or were treated with 20 ng/ml CSF-1 for 5 minutes as described in Materials and Methods. Cells were then fixed and stained for F-actin using Alexa Fluor 568 phalloidin, and for WAVE2, using a polyclonal goat antibody followed by Alexa Fluor 488 anti-goat IgG. Bars, 10 μm.

Interfering with WAVE2 function inhibits CSF-1-induced F-actin rich membrane protrusions. (A) RAW/LR5 cells, transiently transfected with the indicated constructs, were treated with 20 ng/ml CSF-1 for 5 minutes and F-actin-rich protrusions were visualized by Alexa Fluor 568 phalloidin staining. Cells expressing the constructs were identified by epitope staining (FLAG for WAVE1/2 and Myc for WASP) and the number of CSF-1-elicited protrusions was quantified as described in Materials and Methods and expressed as a percentage of the CSF-1 stimulation observed in non transfected cells on the same coverslip; n=3, ^*P<0.05 compared to non transfected cells (represented by the dotted line in A-D). (B) RAW/LR5 cells were transiently transfected with the same constructs as in A, and their ability to undergo Fcγ-R-mediated phagocytosis was determined as described in Materials and Methods and expressed as a percentage of phagocytosis observed in non transfected cells on the same cover slip; n=3, ^*P<0.05 compared to non transfected cells. (C) WAVE1 or WAVE2 antibodies or control IgG were introduced into BMM cells by transient permeabilization as described in Materials and Methods. The ability of cells to form F-actin rich protrusions in response to CSF-1 was analyzed as in A; n=3, ^*P<0.05 compared to non permeabilized cells on the same coverslip. (D) WAVE2 antibodies or control IgG were introduced into RAW/LR5 cells by transient permeabilization, as in C, and CSF-1-induced protrusions were scored as in A; n=3, ^*P<0.05 compared to non permeabilized cells on the same cover slip. (E) WAVE2 and β-actin expression in WAVE2 shRNA-treated RAW/LR5 cells (heterogenous cell populations) was analyzed by western blotting with the appropriate antibodies and compared to mock shRNA treated cells (see inset) and WAVE2/β-actin signal intensity ratios of the indicated blot were quantified. (F) Mock, WAVE2 shRNA-treated cells (white bars) or WAVE2 shRNA-treated cells transiently transfected with a human GFP-WAVE2 construct (black bars) were stimulated with CSF-1 and their ability to form F-actin-rich protrusions in response to CSF-1 was analyzed as in A and expressed as a percentage of the CSF-1 stimulation observed in mock shRNA-treated cells; n=3, ^*P<0.05 compared to mock shRNA-treated cells.

WAVE2 and Abi1 are found in the same complex and are enriched at CSF-1-induced membrane protrusions. (A) FLAG-tagged WAVE2-expressing RAW/LR5 cells were lysed and lysates were sequentially incubated with control IgG and specific antibodies against FLAG for immunoprecipitation (IP). Immunoprecipitates were then subjected to western blotting using the indicated antibodies. Signals corresponding to IgG heavy chain are shown as a proof of equal loading. (B) RAW/LR5 cells were either left untreated (–) or were treated (+) with 20 ng/ml CSF-1 for 5 minutes prior to lysis and immunoprecipitation of Abi1 followed by WAVE2 and Abi1 western blotting. (C) BMM and RAW/LR5 cells were treated with CSF-1, then fixed and stained for WAVE2 and Abi1 as described in Materials in Methods. Bars, 10 μm.

Abi1 and WAVE2 are required for CSF-1-induced F-actin rich membrane protrusions in macrophages. (A) Abi1, WAVE2 and β-actin expression in Abi1 shRNA-treated cells (two different clonal populations shown) was analyzed by western blotting with the appropriate antibodies and compared to mock shRNA-treated cells. Quantification of Abi1/β-actin and WAVE2/β-actin signal intensity ratios is shown below. (B) Mock- and Abi1-shRNA-treated cells were fixed and stained for Abi1 as described in Materials and Methods. Bar, 10 μm. (C) Mock shRNA-treated cells and two independent clones with reduced Abi1/WAVE2 expression (Abi1 shRNA) were treated with 20 ng/ml CSF-1 for 5 minutes and the ability of cells to form F-actin-rich membrane protrusions in response to CSF-1 was scored as described in Fig. 3 and expressed as a percentage of the CSF-1 stimulation observed in mock shRNA-treated cells; n=3, ^*P<0.05 compared to mock shRNA-treated cells.