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First published online 15 January 2008
doi: 10.1242/jcs.010272

Journal of Cell Science 121, 379-390 (2008)
Published by The Company of Biologists 2008
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Membrane targeting of WAVE2 is not sufficient for WAVE2-dependent actin polymerization: a role for IRSp53 in mediating the interaction between Rac and WAVE2

Wassim Abou-Kheir1, Beth Isaac1, Hideki Yamaguchi1,* and Dianne Cox1,2,{ddagger}

1 Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA
2 Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA


Figure 1
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  		Fig. 1. The localization of IRSp53 in macrophages. 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-coupled anti-goat IgG. Bar, 10 µm.

 

Figure 2
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  		Fig. 2. IRSp53 is required for generation of CSF-1-induced F-actin rich membrane protrusions and cell migration. (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 with mock-shRNA-treated cells. A representative blot and quantification of IRSp53:β-actin signal-intensity ratios are shown; n=3; *, P<0.05 compared with 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 with mock-shRNA-treated cells. Representative images and quantification of the IRSp53 signal intensity are shown; n=3; *, P<0.05 compared with 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. 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 a percentage of the CSF-1 stimulation observed in mock-shRNA-treated cells; n=3; *, P<0.05 compared with mock-shRNA-treated cells. The dotted line represents basal ruffling. (E) Mock- (white bar) or IRSp53-shRNA-treated (gray bar) RAW/LR5 cells were fixed after treatment with, or without, CSF-1, and the total F-actin content, normalized to the cell number, was quantitatively measured as described in Materials and Methods and compared with unstimulated control cells; n=3; *, P<0.05 compared with mock-shRNA-treated cells. (F) Chemotaxis and chemokinesis in response to CSF-1 in mock- (white bars) or IRSp53-shRNA-treated (gray bars) RAW/LR5 cells were evaluated using a transmigration chamber assay as described in Materials and Methods. The CSF-1-stimulated migration of each cell population was compared with the corresponding unstimulated condition and expressed as a fold induction. n=3; *, P<0.05 compared with mock-shRNA-treated cells.

 

Figure 3
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  		Fig. 3. IRSp53 is involved in Rac1-mediated ruffling but not Cdc42-mediated formation of filopodia in macrophages. (A) Representative images of F-actin-stained mock- or IRSp53-shRNA-treated nontransfected (top panel), or transfected to express 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. Bar, 10 µm. The ability of mock- (white bars) or IRSp53-shRNA-treated (gray bars) cells to exhibit either ruffles following Rac1Q61L expression (B) or filopodia following Cdc42V12 expression (C) was quantified and expressed as a percentage of the total cells counted; n=3; *, P<0.05 compared with mock-shRNA-treated cells. Bar, 10 µm.

 

Figure 4
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  		Fig. 4. IRSp53 exists in a complex with WAVE2 and Abi1 in a Rac1-dependent manner. (A) Lysates from nontransfected or GFP-tagged IRSp53 and Myc-tagged Rac1- or Rac1N17- or Rac1Q61L- or Cdc42V12-coexpressing RAW/LR5 cells and Cos7 cells were immunoprecipitated with antibodies against either GFP or Myc (IP) and subjected to western blotting using the indicated antibodies. A representative example of three independent experiments is shown. (B) Lysates from mock- or IRSp53-shRNA-treated RAW/LR5 cells expressing Myc-tagged Rac1Q61L were immunoprecipitated with antibodies against Myc (IP) and were then subjected to western blotting using the indicated antibodies. (C) Quantification of WAVE2 or Abi1 co-immunoprecipitated by Myc (from B) is shown; n=3; *, P<0.05 compared with mock-shRNA-treated cells expressing Rac1Q61L. (D) Lysates from nontransfected or Myc-tagged Rac1Q61L- and HA-tagged IRSp53{Delta}SH3-coexpressing 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 Fig. 2D and expressed as a percentage of the CSF-1 stimulation observed in nontransfected mock-shRNA-treated cells; n=3; *, P<0.05 compared with nontransfected mock-shRNA-treated cells (represented by the dotted line).

 

Figure 5
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  		Fig. 5. Membrane targeting of WAVE2 is not sufficient for its regulation. (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. 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 with nontransfected 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 Fig. 2D and expressed as a percentage of the CSF-1 stimulation observed in nontransfected cells on the same coverslip; n=3. (D) RAW/LR5 (white bars) and Cos-7 (gray bars) cells were either transfected with Myc-tagged Rac1Q61L (plain) or RacQ61L and FLAG-tagged WAVE2CAAX (striped) constructs. Coexpressing cells were identified by the Myc and FLAG staining, and the ability of cells to exhibit ruffles was quantified and expressed as a percentage of the 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 Fig. 2D and expressed as a percentage of the CSF-1 stimulation observed in nontransfected mock-shRNA-treated cells; n=3; *, P<0.05 compared with nontransfected mock-shRNA-treated cells (represented by the dotted line).

 

Figure 6
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  		Fig. 6. A model for WAVE2 interaction and regulation in macrophages. (A) Based on several reports, a model for the regulation of WAVE2 has been proposed whereby 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 translocation of WAVE2 to the leading edge, where it stimulates the site-restricted actin polymerization necessary for cell protrusion and motility. (B) Alternatively, the stable WAVE2-Abi1 complex is inactive in the cytoplasm in vivo. Upon stimulation of the CSF-1 receptor (CSF-1R) by CSF-1, activated Rac1 binds to WAVE2, mediated by IRSp53, and induces WAVE2 activation and translocation to the leading edge, where it stimulates the site-restricted actin polymerization required for cell protrusion and motility. Abbreviations: A, acidic region; B, basic region; C, cofilin-homology domain; Pro-rich, proline-rich region; V, verprolin-homology domain; WHD, WAVE-homology domain.

 

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© The Company of Biologists Ltd 2008