WAVE2 deficiency reveals distinct roles in embryogenesis and Rac-mediated actin-based motility
2003; Springer Nature; Volume: 22; Issue: 14 Linguagem: Inglês
10.1093/emboj/cdg350
ISSN1460-2075
Autores Tópico(s)Pluripotent Stem Cells Research
ResumoArticle15 July 2003free access WAVE2 deficiency reveals distinct roles in embryogenesis and Rac-mediated actin-based motility Catherine Yan Catherine Yan Center for Blood Research, 200 Longwood Avenue, Boston, MA, 02115 USA Department of Medicine, Children's Hospital, Boston, MA, 02115 USA Department of Genetics, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Narcisa Martinez-Quiles Narcisa Martinez-Quiles Division of Immunology, Children's Hospital, Boston, MA, 02115 USA Department of Pediatrics, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Sharon Eden Sharon Eden Department of Cell Biology, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Tomoyuki Shibata Tomoyuki Shibata Department of Medicine, Harvard Medical School, Boston, MA, 02115 USA Gastrointestinal Unit (Medical Services) and the Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, 55 Fruit Street, Boston, MA, 02115 USA Search for more papers by this author Fuminao Takeshima Fuminao Takeshima Department of Medicine, Harvard Medical School, Boston, MA, 02115 USA Gastrointestinal Unit (Medical Services) and the Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, 55 Fruit Street, Boston, MA, 02115 USA Search for more papers by this author Reiko Shinkura Reiko Shinkura Howard Hughes Medical Institute, Children's Hospital, Boston, MA, 02115 USA Search for more papers by this author Yuko Fujiwara Yuko Fujiwara Howard Hughes Medical Institute, Children's Hospital, Boston, MA, 02115 USA Division of Hematology and Oncology, Children's Hospital, Boston, MA, 02115 USA Search for more papers by this author Roderick Bronson Roderick Bronson Department of Pathology, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Scott B. Snapper Scott B. Snapper Department of Medicine, Harvard Medical School, Boston, MA, 02115 USA Gastrointestinal Unit (Medical Services) and the Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, 55 Fruit Street, Boston, MA, 02115 USA Search for more papers by this author Marc W. Kirschner Marc W. Kirschner Department of Cell Biology, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Raif Geha Raif Geha Division of Immunology, Children's Hospital, Boston, MA, 02115 USA Department of Pediatrics, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Fred S. Rosen Fred S. Rosen Center for Blood Research, 200 Longwood Avenue, Boston, MA, 02115 USA Department of Medicine, Children's Hospital, Boston, MA, 02115 USA Department of Pediatrics, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Frederick W. Alt Corresponding Author Frederick W. Alt Center for Blood Research, 200 Longwood Avenue, Boston, MA, 02115 USA Department of Medicine, Children's Hospital, Boston, MA, 02115 USA Department of Genetics, Harvard Medical School, Boston, MA, 02115 USA Department of Pediatrics, Harvard Medical School, Boston, MA, 02115 USA Howard Hughes Medical Institute, Children's Hospital, Boston, MA, 02115 USA Search for more papers by this author Catherine Yan Catherine Yan Center for Blood Research, 200 Longwood Avenue, Boston, MA, 02115 USA Department of Medicine, Children's Hospital, Boston, MA, 02115 USA Department of Genetics, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Narcisa Martinez-Quiles Narcisa Martinez-Quiles Division of Immunology, Children's Hospital, Boston, MA, 02115 USA Department of Pediatrics, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Sharon Eden Sharon Eden Department of Cell Biology, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Tomoyuki Shibata Tomoyuki Shibata Department of Medicine, Harvard Medical School, Boston, MA, 02115 USA Gastrointestinal Unit (Medical Services) and the Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, 55 Fruit Street, Boston, MA, 02115 USA Search for more papers by this author Fuminao Takeshima Fuminao Takeshima Department of Medicine, Harvard Medical School, Boston, MA, 02115 USA Gastrointestinal Unit (Medical Services) and the Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, 55 Fruit Street, Boston, MA, 02115 USA Search for more papers by this author Reiko Shinkura Reiko Shinkura Howard Hughes Medical Institute, Children's Hospital, Boston, MA, 02115 USA Search for more papers by this author Yuko Fujiwara Yuko Fujiwara Howard Hughes Medical Institute, Children's Hospital, Boston, MA, 02115 USA Division of Hematology and Oncology, Children's Hospital, Boston, MA, 02115 USA Search for more papers by this author Roderick Bronson Roderick Bronson Department of Pathology, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Scott B. Snapper Scott B. Snapper Department of Medicine, Harvard Medical School, Boston, MA, 02115 USA Gastrointestinal Unit (Medical Services) and the Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, 55 Fruit Street, Boston, MA, 02115 USA Search for more papers by this author Marc W. Kirschner Marc W. Kirschner Department of Cell Biology, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Raif Geha Raif Geha Division of Immunology, Children's Hospital, Boston, MA, 02115 USA Department of Pediatrics, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Fred S. Rosen Fred S. Rosen Center for Blood Research, 200 Longwood Avenue, Boston, MA, 02115 USA Department of Medicine, Children's Hospital, Boston, MA, 02115 USA Department of Pediatrics, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Frederick W. Alt Corresponding Author Frederick W. Alt Center for Blood Research, 200 Longwood Avenue, Boston, MA, 02115 USA Department of Medicine, Children's Hospital, Boston, MA, 02115 USA Department of Genetics, Harvard Medical School, Boston, MA, 02115 USA Department of Pediatrics, Harvard Medical School, Boston, MA, 02115 USA Howard Hughes Medical Institute, Children's Hospital, Boston, MA, 02115 USA Search for more papers by this author Author Information Catherine Yan1,2,3, Narcisa Martinez-Quiles4,5, Sharon Eden6, Tomoyuki Shibata7,8, Fuminao Takeshima7,8, Reiko Shinkura9, Yuko Fujiwara9,10, Roderick Bronson11, Scott B. Snapper7,8, Marc W. Kirschner6, Raif Geha4,5, Fred S. Rosen1,2,5 and Frederick W. Alt 1,2,3,5,9 1Center for Blood Research, 200 Longwood Avenue, Boston, MA, 02115 USA 2Department of Medicine, Children's Hospital, Boston, MA, 02115 USA 3Department of Genetics, Harvard Medical School, Boston, MA, 02115 USA 4Division of Immunology, Children's Hospital, Boston, MA, 02115 USA 5Department of Pediatrics, Harvard Medical School, Boston, MA, 02115 USA 6Department of Cell Biology, Harvard Medical School, Boston, MA, 02115 USA 7Department of Medicine, Harvard Medical School, Boston, MA, 02115 USA 8Gastrointestinal Unit (Medical Services) and the Center for the Study of Inflammatory Bowel Disease, Massachusetts General Hospital, 55 Fruit Street, Boston, MA, 02115 USA 9Howard Hughes Medical Institute, Children's Hospital, Boston, MA, 02115 USA 10Division of Hematology and Oncology, Children's Hospital, Boston, MA, 02115 USA 11Department of Pathology, Harvard Medical School, Boston, MA, 02115 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:3602-3612https://doi.org/10.1093/emboj/cdg350 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Wiskott–Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin-cytoskeletal reorganization downstream of the small Rho GTPase, Rac. We inactivated the WAVE2 gene by gene-targeted mutation to examine its role in murine development and in actin assembly. WAVE2-deficient embryos survived until approximately embryonic day 12.5 and displayed growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2-deficient embryonic stem cells displayed normal proliferation, whereas WAVE2-deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization. These results imply a non-redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin-based processes downstream of Rac that are essential for cell movement. Introduction Actin polymerization is required for diverse cellular processes, including cell growth, motility and polarization. The transduction of extracellular stimuli to reorganize cortical actin filaments is governed by small Rho GTPases, Cdc42 and Rac, which stimulate polarized forward protrusions (filopodia and lamellipodia) at the leading edge of cells, and RhoA, which induces retractions at the tail end of cells. The Wiskott–Aldrich syndrome protein (WASP) family, referred to as WASPs (WASP and N-WASP) and SCAR/WAVEs (WAVE1, WAVE2 and WAVE3), are cytoplasmic molecules that link Rho GTPases and actin assembly (Suetsugu et al., 1999; Takenawa and Miki, 2001). In response to activation of small Rho GTPases, these proteins stimulate the nucleating activity of the Arp2/3 complex, resulting in specific cell surface projections: filopodia by N-WASP following activation by Cdc42, and lamellipodia by WAVE molecules following activation by Rac (Miki et al., 1998a,b; Takenawa and Miki, 2001). One SCAR/WAVE protein exists in Dictyostelium discoideum, Caenorhabditis elegans and Drosophila melanogaster, and three orthologs (WAVE1, WAVE2 and WAVE3) are conserved in mammals (Bear et al., 1998; Suetsugu et al., 1999; Benachenhou et al., 2002). WAVE1 and WAVE3 are predominantly expressed in the brain; like N-WASP, WAVE2 is widely expressed (Bear et al., 1998; Suetsugu et al., 1999; Benachenhou et al., 2002; Soderling et al., 2003). SCAR/WAVE proteins were first identified in Dictyostelium as negative regulators of G protein coupled signaling (Bear et al., 1998) and in transient assays as a downstream effector for Rac- mediated lamellipodia formation (Miki et al., 1998b). In Dictyostelium, scar-deficient cells generated by homologous recombination display aberrant morphology and actin distribution during chemotaxis, reduced F-actin staining during vegetative growth, and reduced fluid phase influx and endocytic trafficking (Bear et al., 1998; Seastone et al., 2001). In Drosophila, inactivation of the single SCAR/WAVE gene shows the encoded protein plays the major role in Arp2/3-dependent regulation of cell morphology, whereas WASP appears to be largely dispensable (Zallen et al., 2002). The conserved Verprolin-cofilin-acidic (VCA)-rich domain in both WASPs and WAVEs mediates binding and activation of Arp2/3 catalysis of actin polymerization (Machesky and Insall, 1998; Miki et al., 1998b; Suetsugu et al., 1999; Zalevsky et al., 2001). In contrast to WASPs, WAVEs share a common SCAR homology domain and are missing the Cdc42/Rac interactive binding (CRIB) domain that mediates WASPs interaction and activation by Rho GTPases (Suetsugu et al., 1999; Kim et al., 2000). The mechanism of regulation and activation of WASPs and WAVEs are different. In the case of WASPs, the VCA domain is autoinhibited by intramolecular interactions with the CRIB domain (Kim et al., 2000) and activated by interactions with GTP–Cdc42 and phosphatidylinositol-4,5-biphosphate. In contrast, the full-length recombinant WAVE1 protein is constitutively active (Machesky et al., 1999), whereas in vivo it is maintained in an inactive heteropentameric complex that includes WAVE, the p53-inducible PIR121, the Nck-associated protein Nap125, the Abl interactor protein Abi2 and a small actin-stimulating peptide, HSPC300 (Eden et al., 2002). Activated Rac or the Src homology 3 (SH3)–SH2 adaptor protein Nck dissociates the WAVE complex to release the active WAVE–HSPC300 complex to promote actin nucleation (Cory and Ridley, 2002; Eden et al., 2002). Rac signaling to actin is then proposed to be attenuated by recruitment of the Rac-selective GTPase activating protein WRP by WAVE1, which hydrolyzes Rac to the inactive GDP bound form (Soderling et al., 2002). Currently, the rapidly expanding list of WAVE protein partners includes cytoskeletal components (profilin, α-tubulin and spectrin αII and βIII) and SH3 proteins involved in signal transduction linked to WASPs and Rho GTPase, Ras and Rab GTPase pathways (Miki et al., 1998b, 1999, 2000; Pollard et al., 2000; Westphal et al., 2000; Nakagawa et al., 2001; Soderling et al., 2002; Sun et al., 2003). Here, we have focused our studies on WAVE2, the most widely expressed and leukocyte-dominant WAVE isoform. To directly assess the physiological consequences of a WAVE2 deficiency, we inactivated the WAVE2 gene in the mouse germline to examine its role in murine development and generated WAVE2-deficient cell lines to examine its role in actin-based cell motility. Results Gene-targeted inactivation of WAVE2 We used RT–PCR to obtain a WAVE2 cDNA from murine 129sv/ev splenocytes. Human and murine WAVE2 protein sequences share 92.6% identity. We determined that murine WAVE2 RNA, like human WAVE2 RNA, is widely expressed (Suetsugu et al., 1999). We mapped the murine WAVE2 genomic locus from a murine 129sv phage library (Stratagene). The WAVE2 gene is encoded by eight exons spanning 20 kb of mouse chromosome 4. To abolish WAVE2 function, we replaced the sequences spanning exons 3–7 with a neomycin-resistance gene (neor) flanked by loxP sites in TC1 embryonic stem (ES) cells (Figure 1A). An IRES–EGFP (intra-ribosomal entry sequence and enhanced GFP) was inserted into intron 2 to allow for the potential assessment of the developmental regulation of the WAVE2 gene upon Cre-mediated excision of the neor marker in vivo. We used two independent WAVE2+/neo targeted ES cell lines to generate mice carrying the WAVE2-replacement mutation in the germline (WAVE2+/neo mice) (Figure 1B, Tail southern). To confirm that the targeted replacement of exons 3–7 resulted in loss of WAVE2 RNA and protein expression (Figure 1C), we generated WAVE2neo/neo ES cells by increasing G418 drug selection (data not shown) or by sequential targeting of the wild-type (WT) allele in WAVE2+/− ES cells after Cre-mediated excision of the loxP-flanked neor cassette (Figure 1B, ES targetings, −/neo lane). Immunoblot analyses of WAVE2neo/neo, WAVE2−/− ES cells, WAVE2-deficient ES-derived fibroblast-like cells (Figure 1C; data not shown) and WAVE2-deficient mouse embryonic fibroblasts (MEFs; shown below in Figure 5A) all failed to detect WAVE2 protein expression. As we were able to generate WAVE2-deficient ES cell lines, we can conclude that WAVE2 is not required for the survival of ES cells. Figure 1.Murine WAVE2 RNA expression and targeted disruption of WAVE2 in mice and cells. (A) Diagram of murine WAVE2 genomic locus and targeting strategy. The physical map of the entire WAVE2 genomic locus containing all eight exons is shown. Exons are indicated by small rectangular boxes. Genomic DNA fragments used to generate the targeting construct are hatched. The neomycin-resistance (Neor) and thymidine kinase (TK) genes used for positive and negative selections, the 5′ and 3′ probes, and the Xba1 restriction sites used for screening embryonic stem (ES) cell transfectants by Southern blot analyses are labeled. Deletion of the loxP-flanked neor cassette leaves a single loxP site and the GFP downstream of an intra-ribosomal entry sequence (IRES). (B) Generation of WAVE2+/neo, WAVE2neo/neo and WAVE2−/neo ES cells and germline mice. Using the 5′ probe noted in the schematic in (A), homologous recombination results in the loss of a single 7.6 kb band and the appearance of a 8.6 kb targeted band or a 7 kb Cre deleted band. Genotypes of germline WAVE2+/neo mice born from mating a WAVE2 chimeric male and a 129sv/ev wild-type female are shown. (C) Loss of WAVE2 protein and RNA expression in WAVE2-deficient ES cells. Western blot analyses of protein extracts from wild-type (+/+), WAVE2+/neo (+/neo) and WAVE2neo/neo (neo/neo) ES cells. WAVE2 runs at the 84 kDa molecular weight marker and is absent in WAVE2-deficient ES cells. Equal protein loading was determined using an antibody to Ku86 (Santa Cruz). Northern blot analysis was determined using a WAVE2 cDNA probe. Equal RNA loading was determined with a β-actin control probe. Download figure Download PowerPoint WAVE2-deficient embryos have gross morphological defects but normal organogenesis Mating of WAVE2+/neo male and female mice yielded live WAVE2+/+ and WAVE2+/neo pups at the expected Mendelian ratios with no obvious defects in weight or reproductive vigor of the mice. As these breedings failed to yield WAVE2neo/neo progeny (Table I), we performed timed mating and analyzed embryos at various gestational stages to determine when embryonic lethality occurs. Through examination of embryos obtained from embryonic day 7.5 (E7.5) to E17 (confirmed by Southern blot analyses), we determined that WAVE2neo/neo embryos are able to survive through E12–E12.5. Gross examination of dissected WAVE2-deficient embryos shows that they display mild to severe developmental growth retardations (Figure 2A–C). All WAVE2neo/neo embryos showed exterior morphological abnormalities of head and facial structures and posterior limbs and variable abnormalities in body conformation (Figure 2A–C). Figure 2.WAVE2 deficiency results in mild to severe growth retardation and profound morphological defects in embryos. (A–C) Micrographs of E11–E12 wild-type (wt), WAVE2+/neo (Het) and WAVE2neo/neo (mut) embryos derived from F1 WAVE+/neo matings. Embryo littermates were photographed together at 20× (B and C) or 40× (A) magnification. The micrograph in (A) shows embryos fixed in Bouin's Solution (Sigma); (B) and (C) show micrographs of unfixed embryos. Distinct growth retardations of mut versus wt embryos are apparent in (A) and (B). Branchial arches, limb buds, somites (A, B and C) and blood (B and C) are apparent. In (C), the mut embryo is comparable in size to wt and Het littermates but displays distinct conformational abnormalities: decreased size and malformation of cephalic (head) and caudal extremities and increased hemorrhaging. (D–I) High-powered hematoxylin-eosin staining of the transverse sections of WAVE2+/neo (Het) and WAVE2neo/neo (mut) embryo littermates. (D, E, F and G) Transverse section of 6–7 μm thickness taken from the cephalic region of WAVE2+/neo (D and F) and WAVE2neo/neo (E and G). The forebrain (fb) is oriented at the top and the hindbrain (hb) at the bottom. More anterior section (D and E) displaying the three ventricles of the forebrain (fb) and later sections (F and G) displaying lens and cheek formation are shown. Significant malformations of ventricles in both the forebrain and hindbrain are apparent. (H and I) Transverse section of WAVE2+/neo (H) and WAVE2neo/neo (I) caudal somites. Arrows point to two of several regions that display unusual detachment of cells. Download figure Download PowerPoint Table 1. WAVE2 deficiency results in embryonic lethality during mid-gestation Gestational agea +/+ +/neo neo/neo Resorbed Total Neonate/birth 45 81 0 nd 126 >E13 17 23 0 10 50 E12–E12.5 2 4 1 2 9 E11.5–E12 11 18 10 3 42 E9.5–E11.5 9 16 11 0 36 E7.5–E8.5 5 14 7 0 26 Gestational ageb +/+ +/− −/− Resorbed Total Neonate/birth 23 51 0 nd 74 Live pups and embryos were genotypes by Southern blot analyses of yolk sac or tissue from embryos. a Offspring from WAVE2 matings; b Offspring from WAVE2 matings; nd, not determined. Histological analyses of these embryos revealed prominent morphological abnormalities in the appearance of the forebrain and hindbrain ventricles (Figure 2; compare D and F with E and G), reduced ventricle length, and disruption in the cellular organization of caudal somites, the mesodermal tissue which gives rise to muscle, the spinal column and the dermis (Figure 2; compare H and I). As we did not observe an increase in apoptotic cells, the decreased ventricle length and cellular complexity of the caudal somites cannot be attributed to increased cell death (data not shown). In some instances, we also observed hemorrhaging throughout the embryo body (Figure 2C, mut). Despite developmental delays, there was no apparent neural tube or cardiac abnormalities as observed for a deficiency in N-WASP (Snapper et al., 2001). In all the embryos examined, we observed normal organogenesis of the fetal liver and heart and normal extra-embryonic placental tissue when compared with WAVE2+/+ and WAVE2+/neo littermates (data not shown). Therefore, the exact cause of embryonic lethality, although undetermined, cannot be attributed to a placental defect. The observed embryonic lethality resulting from WAVE2 deficiency is also not due to cis effects from insertion of the PGK-neor cassette on neighboring genes. We generated WAVE2+/− mice by crossing WAVE+/neo mice to Cre-expressing EIIA-Cre/CD1 mice, which delete during the zygotic stage of embryonic development (Lakso et al., 1996). Interbreeding of the resulting WAVE2+/− mice did not give rise to viable WAVE2−/− pups (Table I). Analyses of MEFs isolated from E9.5 and E11 WAVE2neo/neo knockout (KO) embryos indicated a striking reduction in proliferation when compared with WAVE2+/neo (Het) and WAVE2+/+ wild-type (WT) MEFs (Figure 3A), whereas WAVE2neo/neo and WAVE2−/− ES cells grew normally (Figure 3B). The proliferation of WAVE2-deficient MEFs was partially restored by ectopic expression of a WAVE2-expressing retrovirus (Figure 3A, RVW2). This partial versus a full restoration of growth may reflect factors including the efficiency of transduction of the WAVE2 retrovirus (∼30%) and differential regulation of the ectopic WAVE2 protein expression in the KO MEFs. We conclude that, although WAVE2 is not required for the proliferation and survival of ES cells, it is required for the normal proliferation of MEFs. Figure 3.Growth rate of WAVE2-deficient cell lines. (A) Comparison of the growth rate of primary mouse embryonic fibroblasts (MEFs) derived from E11 embryo littermates, one wild-type (376-3+/+), two WAVE2+/neo (376-5+/− and 376-9+/−) and three WAVE2neo/neo MEFs (376-1−/−, 376-6−/− and 376-8−/−), and the growth rates of primary MEFs, untransduced (44−/− and 61-12−/−) or transduced with retrovirus expressing GFP (WTRVGFP and 44−/−RVGFP) or the WAVE2–GFP (61-12−/−RVW2 and 44−/−RVW2). Early passage cells were plated at 2 × 104 per well in a 6-well dish in triplicate fashion. Cells were counted every 2 days up to 9 days in two independent experiments. The average of each experimental point with standard error bars is shown (hatched bars). Additional growth curves of primary MEFs derived from E9.5 embryo littermates gave similar results (data not shown). (B) The growth rate of WAVE2-deficient (KO) embryonic stem (ES) cells containing or lacking the loxP-flanked neor cassette are compared with wild-type (WT) and two Het ES cell lines. The genotypes of the ES cell lines are depicted. ES cells, depleted of MEFs, were plated at 2 × 104 cells per well in a 6-well dish and counted in triplicate as described above. The average of each experimental point with standard error bars is shown (hatched bars). (C) Flow cytometric analyses of FITC-Annexin V binding versus Topro 3 uptake in WT versus KO MEFs. Upper left, damaged cells (Annexin V−, Topro 3+); lower left, live cells (Annexin V−, Topro 3−); upper right, late apoptotic and dead cells (Annexin V+, Topro 3+); lower right, early apoptotic cells (Annexin V+, Topro 3−). Numbers represent the average of three experiments. (D) WAVE2 KO MEFs contain fewer replicating cells. WT and KO MEFs were pulsed for 1.5 h with 20 μM bromodeoxyuridine (BrdU) and analyzed by flow cytometry. The average percentage of labeled cells among total live cells from three experiments is indicated by the box. (E) Kinetics of BrdU incorporation of WT versus KO MEFs. MEFs were continuously labeled with 100 μM BrdU for 48 h and analyzed by flow cytometry. The average percentage of BrdU-labeled cells among total live cells from three experiments with standard error bars is plotted at each time point. Download figure Download PowerPoint We further determined that the slower progression of KO MEFs is not due to an increase in apoptosis, as measured by Annexin V staining of cells (Figure 3C), whereas the percentage of replicating cells in asynchronous MEF populations measured by bromodeoxyuridine (BrdU) incorporation appeared to be significantly reduced (Figure 3D and E). Compared with WT MEFs, 70% fewer KO MEFs were in S phase, indicating that at any given time KO MEFs contained fewer replicating cells (Figure 3D). To track the kinetics of S phase entry of total asynchronous MEF populations, WT and KO MEFs were continuously labeled with BrdU-containing media and sampled for up to 48 h (Figure 3E). KO MEFs consistently displayed markedly fewer cells in S phase than WT MEFs. At 48 h, >89% of WT MEFs incorporated BrdU, compared with <43% of KO MEFs. We conclude that the reduced proliferation of KO MEFs is associated primarily with slower cell cycle progression. The significant decrease in growth rate of WAVE2 KO MEFs led us to establish fibroblast cell lines by introducing retrovirus that expresses the human papilloma virus E6 and E7 proteins into these MEFs. Transduction of the E6 and E7 proteins into primary cells has been shown to promote long-term culturing of cells through inactivation of p53 and Rb (von Knebel Doeberitz et al., 1994; Foster and Galloway, 1996; Furukawa et al., 1996; Spitkovsky et al., 2002). Independent fibroblast cell lines of each genotype derived from the MEFs (Figure 3) were established. As has been previously reported for other cell types (von Knebel Doeberitz et al., 1994; Furukawa et al., 1996; Spitkovsky et al., 2002), transduction of the E6/E7 retrovirus also changed proliferation capacity of the KO fibroblasts, such that they grew normally, comparable with WT and Het fibroblast lines (data not shown). However, their responses to growth factor stimulation and Rac activation (described below) remain similar to that of primary KO MEFs. WAVE2-deficient MEFs display defects in actin-cytoskeletal formations WAVE2 has been implicated in lamellipodium formation downstream of the small GTPase Rac (Miki et al., 1998b), which is in turn activated by various growth factors, such as platelet-derived growth factor (PDGF) (Bishop and Hall, 2000). To gain insights into the function of WAVE2 protein in actin-cytoskeletal regulation, primary MEFs generated from WAVE2neo/neo embryos were used to characterize the role of WAVE2 in actin-cytoskeletal organization (Figure 4). WT and WAVE2 KO MEFS were serum starved overnight (in 1% FCS) and then stimulated with PDGF (20 ng/ml) for 10 min (Figure 4A). Quiescent (unstimulated) or PDGF-stimulated cells were stained with TRITC-phalloidin to detect the actin cytoskeleton (red panels) and immunostained with antibody to cortactin to detect membrane ruffles (green panels), represented by circular membrane ruffles (blue arrows) or lamellipodia (yellow arrows) (Figure 4A). Cortactin, a protein implicated in lamellipodium formation downstream of both PDGF and Rac (Weed et al., 1998), localizes specifically to membrane ruffles, including circular membrane ruffles and lamellipodia, giving us a sensitive method to differentiate between the different ruffling capabilities of all cells examined in our assay. We observed continuous staining of cortactin and actin along the border of WT MEFs, termed the leading edge, that was predominantly either missing or irregularly dispersed at tips of membrane protrusions in KO MEFs (Figure 4A; compare WT and KO, indicated by yellow arrows). Figure 4.WAVE2 is essential for lamellipodium and ruffle formation in mouse embryonic fibroblasts (MEFs). (A) Sub-confluent serum-starved wild-type (WT) and WAVE2 knockout (KO) MEFs were left unstimulated or stimulated with 20 ng/ml PDGF for 10 min. The leading edge and other areas of lamellipodia and ruffles are more obvious by cortactin staining (green panels) and detection of F-actin cytoskeleton by TRITC-phalloidin (red panels). KO cells show more distinct cytoskeletal defects. Note the absence of leading edge and aberrant lamellipodia formation (yellow arrows point to leading edge of lamellipodia) in unstimulated KO cells compared with WT cells. PDGF induces lamellipodia (yellow arrows), and circular ruffles (blue arrows) in KO MEFs are structurally malformed. Scale bar, 10 μm. (B) WAVE2 retroviral expression rescues the defect in lamellipodium formation. WAVE2 KO MEFs transduced with WAVE2 retrovirus (RVWAVE2), left unstimulated or stimulated with 20 ng/ml PDGF for 10 min. Cortactin staining is shown in red and F-actin in blue. Rescued KO MEFs exhibit a normal morphology with well-defined lamellipodium areas. (C) Membrane ruffle formation of WT, KO and Rescue (KO MEFs rescued with the WAVE2 retrovirus). One hundred cells of each condition were counted. Membrane ruffles are broken down into two distinct categories: lamellipodia and circular ruf
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