Essential role for Abi1 in embryonic survival and WAVE2 complex integrity

Abstract

Abl interactor 1 (Abi1) plays a critical function in actin cytoskeleton dynamics through participation in the WAVE2 complex. To gain a better understanding of the specific role of Abi1, we generated a conditional Abi1-KO mouse model and MEFs lacking Abi1 expression. Abi1-KO cells displayed defective regulation of the actin cytoskeleton, and this dysregulation was ascribed to altered activity of the WAVE2 complex. Changes in motility of Abi1-KO cells were manifested by a decreased migration rate and distance but increased directional persistence. Although these phenotypes did not correlate with peripheral ruffling, which was unaffected, Abi1-KO cells exhibited decreased dorsal ruffling. Western blotting analysis of Abi1-KO cell lysates indicated reduced levels of the WAVE complex components WAVE1 and WAVE2, Nap1, and Sra-1/PIR121. Although relative Abi2 levels were more than doubled in Abi1-KO cells, the absolute Abi2 expression in these cells amounted only to a fifth of Abi1 levels in the control cell line. This finding suggests that the presence of Abi1 is critical for the integrity and stability of WAVE complex and that Abi2 levels are not sufficiently increased to compensate fully for the loss of Abi1 in KO cells and to restore the integrity and function of the WAVE complex. The essential function of Abi1 in WAVE complexes and their regulation might explain the observed embryonic lethality of Abi1-deficient embryos, which survived until approximately embryonic day 11.5 and displayed malformations in the developing heart and brain. Cells lacking Abi1 and the conditional Abi1-KO mouse will serve as critical models for defining Abi1 function.

Essential physiological processes such as cell migration, adhesion, and endocytosis are dependent on the coordinated remodeling of the actin cytoskeleton, but the complexity of the mechanisms at play is only beginning to be appreciated (1, 2). Nucleation of actin filaments is catalyzed by different types of protein complexes, the most prominent of which include the Arp2/3 complex and members of the formin family of proteins (3). The Arp2/3 complex is regulated by additional proteins called “nucleation-promoting factors” (NPFs). The most prominent NPFs include the WASP and WAVE subfamilies (4), not only because they constitute key signaling nodes that connect activation signals from the Rho family of small GTPases with the actin polymerization machineries but also because the N-WASP and Arp2/3 complexes have emerged as frequent targets for viral or bacterial pathogens that usurp the host actin cytoskeleton for their needs (5).

Abl interactor 1 (Abi1) is one of the major regulators of actin cytoskeleton reorganization through participation in several multiprotein complexes that either associate with actin filaments through spectrin (6) or Eps8 (7) or that regulate the dynamics of actin polymerization more indirectly, for instance through regulation of small GTPase activities of the Rho family (4, 8). Rho GTPases are modulated by complexes of Abi1 with Eps8–Sos1 (9, 10) and downstream actin polymerization e.g. by N-WASP (11) and WAVE complex (12–14). The ubiquitous WAVE complex, comprising the direct Rac interactor Sra-1/PIR121, its binding partner Nap1, plus Abi1, HSPC300/Brick1, and WAVE2, is considered to be the essential functional output module linking Rac1 activation to actin polymerization that drives lamellipodia protrusion at the plasma membrane (13, 14). WAVE1 and WAVE3 were proposed to have roles analogous to WAVE2 in the complex (8). Although the function of WAVE3 is less defined, the critical role of the other two WAVEs in dorsal ruffle formation was demonstrated (15, 16). Mechanistically, it is proposed that native WAVE2 complex is inactive (17–19) but is activated at the membrane by simultaneous interaction with prenylated Rac-GTP and acidic phospholipids as well as by serine/threonine phosphorylation (20). Rac1 does not interact directly with WAVE but instead binds to the Nap1-binding protein Sra-1 (12) and to Abi1 (21). The Abi1-mediated tyrosine phosphorylation of WAVE2 by Abl regulates WAVE2 complex activity either by regulating conformation of the complex, as suggested (22), or by regulating interactions among the complex components or with other cellular targets (10, 23, 24). The interaction of Abi1 with p85 (24) might modulate PI3 kinase activity (10); this activity would be consistent with coincident modulation of WAVE complex activity by its interaction with PIP3, or PIP3-mediated membrane recruitment of WAVE2 (25).

The Xenopus laevis homolog of Abi1, xlan4, was demonstrated to be critical for CNS development, and Abi2 might be similarly required in mice (26). In mice Abi2 has regulatory functions in cell–cell adhesion, cell migration, and tissue morphogenesis (27). Abi2-KO mice exhibit a severe memory loss thought to be caused by deficiencies in dendritic spine and adherens junction formation and abnormal cell–cell communication (27). A role for Abi in regulation of the cytoskeleton and in cytokinesis has been confirmed in Dictyostelium (28). Dictyostelium has only one Abi gene; thus it offers no specific conclusion for roles of mammalian Abi1 or Abi2. Moreover, numerous studies have concluded that Abi proteins have overlapping functions in actin cytoskeleton regulation (29, 30). Studies using conventional Abi1 (31) and WAVE2 (16) mutant alleles suggested a critical role of Abi1 in embryonic development, thus suggesting that a conditional allele would provide more definitive information about Abi1, including its function in adult organs.

Here we present the initial characterization of MEFs isolated from a conditional Abi1-KO mouse. Biochemical analyses of Abi1-KO cells indicated that the presence of Abi1 is critical for WAVE2 complex integrity. As a physiological readout of Abi1 gene disruption, we observed impairment of local actin polymerization processes leading to decreased dorsal ruffle formation and cell migration. We also found that Abi1 is essential for embryonic development, because Abi1-deficient embryos survived only until embryonic day 11.5 (E11.5) and displayed malformations in the developing heart and brain.

Results

Generation of Abi1 Floxed Strain.

To generate the Abi1 conditional knockout mouse, we prepared a targeting vector that would result in conditional deletion of Abi1 exon 1, which is consistent with the alternatively spliced Abi1 mRNA (6) (Fig. 1A). Design of the targeting vector and subsequent steps of cloning of the transgenic animal, including F1 transmission of the transgene and production of the homozygous Abi1 floxed (fl/fl) strain (Fig. 1 B and C), are described in SI Materials and Methods.

Fig. 1.

Generation of conditional Abi1-KO mouse. (A) Design of the targeting vector and subsequent steps of recombinant Abi1 allele modification. Abi1 allele, wild-type Abi1 allele; targeted allele, the targeting vector containing exon 1 with the short homology arm (SA) extending 3′ and the long homology arm (LA) on the 5′ side of exon 1. Exon 1 is flanked by two loxP sites, and the neomycin gene cassette is inserted 3′ to exon 1 and internal to the distal loxP site. The neomycin gene cassette is bound by two frt sites and includes an additional loxP site as indicated (SI Materials and Methods). The target region is 0.9 kb and includes exon 1. Floxed allele, the recombinant Abi1 allele following frt-mediated removal of the neomycin cassette; deleted allele, the recombinant Abi1 allele following Cre-mediated removal of exon 1. (B) PCR analysis of genotypes of parental and F1 mice. Heterozygous Abi1 mice carrying the targeted allele were bred with the frt deleter strain (SI Materials and Methods). Animals were genotyped with primers LAN1 and A2 for the neomycin gene cassette (Neo⁺/frt⁺/loxP⁺), with primers mAbi1loxP35′ and Flankneo13′ (SI Materials and Methods and Fig. S1) for the wild-type allele (Upper panel, lower band) or with the floxed Abi1 allele, which lacks the neomycin gene and the 3′ frt site (Neo⁻/frt⁻/loxP⁺) (Upper panel, upper band). Under the PCR conditions used, no amplification of the neomycin-positive allele (Neo⁺/frt⁺/loxP⁺) was observed with primers mAbi1loxP35′ and Flankneo13′. Animals 1 and 3 are heterozygous mice positive for the floxed allele lacking neomycin and the wild-type allele (Upper); and they lack the neomycin gene (Lower). Animal 2 contains only wild-type alleles (Lower). (C) Southern blot analysis of Abi1 transgenic mice. Southern blotting was performed as described (SI Materials and Methods). Mouse genomic DNA was digested with AflII enzyme and hybridized with a 543-bp PB1/2 probe. Expected sizes were 4.15 kb for the wild-type (wt); 5.49 kb for the targeted allele (loxP⁺/neo⁺), (indicated by stars); and 3.65 kb for the floxed allele with the neomycin cassette deleted (loxP⁺/neo⁻). (D) Phenotype of Abi1-null embryos. Phenotypic analyses show exencephaly and pericardial edema in Abi-null embryos. The table summarizes observations of embryos at days E9.5, E10.5, and E11. Embryo death occurred between E10.5–E11.5.

Absence of Abi1 Causes Embryonic Lethality at Day E11.5 of Development.

It has been shown that Nap1 is essential for a series of actin-mediated cell-migration events during early mouse embryogenesis (32). These events include migration of the anterior visceral endoderm, required for specification of the anterior–posterior body axis of the animal, and migration of the mesoderm and endoderm germ layers (32). As a WAVE complex component, Abi1 is involved in the regulation of cell migration; therefore it was expected that Abi1 also might regulate morphogenetic events during early mouse embryogenesis. By excision of the floxed exon 1 from the germ line, a null allele of the Abi1 gene was obtained. Using animals carrying this null Abi1 allele (SI Materials and Methods), timed matings were carried out, and the phenotypes of the homozygous null embryos from day E8.5 to E11.5 were analyzed. Unlike Nap1 mutations, which cause developmental arrest at day E9.5, Abi1-null embryos displayed lethality between E10.5 and E11.5 (Fig. 1D). Gross examination of E8.0–E11.0 embryos revealed marked developmental delay with prominent neural tube and cardiac abnormalities similar to those observed for N-Wasp–null embryos (33). As early as E8.5, Abi-null embryos exhibited developmental delay (smaller size, some unturned embryos). Undulation of the neural tube was observed, and in some embryos the neural tube was open. Cardiac tissue was clearly apparent in Abi1-null embryos, but at late stages the heart was markedly dilated (cardiac edema). Abi1-null embryos developed somites and limb buds; however, severe abnormalities were observed in the formation of branchial arches. Although the specific developmental abnormalities leading to death of the Abi1-null embryos remain uncertain, defective cardiac function and/or abnormalities in branchial arch formation might be the causes. Nonetheless, some Abi1-null embryos were observed to have a beating heart up to E10.5. No Abi1-null embryos survived beyond E11.5, although lack of Abi1 seemed not to affect gastrulation. The most marked defect was the developmental delay of Abi1-null embryos and the pronounced neural and cardiac defects.

Instability of the WAVE2 Complex in Abi1-KO MEFs.

After isolation and PCR screening of MEF cell clones, Southern blotting confirmed Cre-mediated deletion of exon 1, which resulted in the lack of Abi1 expression (Fig. 2 and Figs. S2 and S3). Using Abi1-KO cells, we investigated how loss of Abi1 affects the stability of other WAVE2 complex subunits. Western blot analysis of total cell extracts demonstrated that WAVE2, Sra-1, and Nap1 protein levels were reduced significantly, although not abolished, in Abi1-KO cells as compared with the Abi1-expressing parental cell line (Fig. 3). A twofold increase in Abi2 protein levels in Abi1-KO vs. control cells also was observed. An anti-WAVE1 antibody mirrored the significant down-regulation observed for WAVE2, but the opposite was observed for WAVE3 (Fig. 3). However, it should be noted that a pan-WAVE antibody capable of recognizing all WAVE proteins still showed reduced WAVE1/2/3 levels in Abi1-KO clones compared with control (Fig. 3 Right and Fig. S4A). This finding is consistent with the observed phenotypes and may indicate that increased WAVE3 levels cannot compensate for the reduction of WAVE1 and WAVE2, probably because of the relatively low abundance of the predominantly neuronal-specific WAVE3 (34) in these cells.

Fig. 2.

Characterization of Abi1-KO MEF cell lines. (A) Cre-recombinase–mediated exon 1 deletion of Abi1 in MEF cell lines. DNA from MEF cells was genotyped with primers DL75′ and Neogene13′ (SI Materials and Methods and Fig. S1). Genotyping of MEF cell lines based on the PCR result is indicated below the panel. Floxed, Abi1-floxed allele; deleted, exon 1-deleted allele; Abi1 (fl/fl), genotype homozygous for the floxed allele; Abi1 (del/del), genotype homozygous for the deleted allele; Abi1 (fl/del), heterozygous genotype. Note that in cell line #8 clone d3 has both alleles, indicating that in this cell line Cre-recombinase failed to recombine on one allele; hence the heterozygous genotype, Abi1 (fl/del), is observed. Primer sequences are listed in SI Materials and Methods. (B) Southern blot analysis of MEF cell genomic DNA. MEF cell genomic DNA was digested with AflII enzyme and hybridized with the 543-bp PB1/2 probe. Expected sizes were 4.15 kb for wild type; 3.65 kb for the floxed allele (neomycin cassette deleted; loxP⁺/neo⁻); and 4.84 kb for the deleted allele [exon 1 and neomycin cassette deleted; loxP⁻/neo⁻(Ex1del)]. (C) Cre-recombinase–mediated loss of Abi1 expression in MEF #3 cell line subclones. Western blot analysis of Abi1 expression in parental MEF #3 Abi1 (fl/fl) and in exon 1-deleted Abi1 (del/del) MEF cell lines. Cell lysates of the indicated cell lines were blotted with antibody 7B6 (23). Clones #3-1 through #3-11 are subclones of the parental MEF#3 obtained following transient Cre-recombinase expression. Abi1(^+/+) MEF WT, mouse embryonic cells expressing the wild-type Abi1 gene. Protein-stained gel of the samples used for Western blot was used as loading control.

Fig. 3.

Expression levels of WAVE proteins, Sra-1, and Nap1 in Abi1-KO cells. Western blot analysis of MEF cell lysates of Abi1-KO control (fl/fl) and Abi1-KO cell lines. WAVE1, -2, -3, Nap1, Sra-1, and Abi2, protein levels were evaluated with specific antibodies in total cellular lysates obtained from two clones [c1 (#3-6) and c2 (#3-11)] lacking Abi1 expression and from the parental line MEF #3. A significant decrease in protein levels of the WAVE-complex components Sra-1 and Nap1, WAVE1, and total WAVE1/2/3 and a significant (twofold) increase in WAVE3 and Abi2 levels were observed in KO cells vs. control. Levels of protein expression ± SEM were evaluated based on band intensities from several independent experiments (Center). Anti-Rac1 antibody (Rac1) was used as loading control. Abi1 was detected with monoclonal antibody 1B9 (SI Materials and Methods).

Increased Levels of Abi2 Expression in an Abi1-KO Cell Line Reached Only 22% of the Endogenous Abi1 Level in the Control Floxed Cell Line.

To determine the absolute concentration of Abi1 proteins in cell lysates, predetermined amounts of purified recombinant Abi1 or Abi2 protein standards were added into MEF cell lysate samples from Abi1-KO and Abi1 floxed (fl/fl) control cell lines. The comparative evaluation of the control recombinant Abi1 and Abi2 (Fig. S5) amounts vs. their endogenous levels, as detected by respective antibodies, allowed determination of the absolute amount of endogenous Abi1 or Abi2 proteins. Standard curves were generated by plotting the band intensities against the concentrations of control recombinant Abi GST-tagged proteins (∼80 kDa) (Fig. S6). Based on the standard curves, we calculated that, although the relative intensity of Abi2 was about two times higher in Abi1-KO cells than in control cells, the difference in the absolute amount of endogenous Abi2 vs. Abi1 in control cells was less than 10%: 0.914 ± 0.096 ng Abi2 vs. 11.88 ± 0.55 ng Abi1 per microgram of total protein in the sample. This evaluation indicated that despite enhancement of Abi2 expression in Abi1-KO cells, its level did not exceed 22% of the endogenous Abi1 in control cells. Thus, it seems likely that the failure to up-regulate Abi2 sufficiently is one of the reasons Abi2 does not reconstitute the WAVE complex in Abi1-KO cell lines.

Defect in Formation of PDGF-Induced Dorsal Ruffles in Abi1-KO MEF Cells.

Abi1, as the critical component of WAVE2 complex, is known to be involved in actin reorganization resulting in lamellipodia and in actin-rich peripheral and dorsal ruffle formation upon growth factor stimulation (9, 30, 35). Therefore, we asked whether any defects in these structures were observed in cells lacking expression of a functional Abi1 gene. We found that upon PDGF treatment, both Abi1 control and Abi1-deficient cells displayed peripheral as well as dorsal ruffle formation, as assessed by Alexa Fluor 594–phalloidin staining, and WAVE2 was localized to these structures, as expected, although it was less prominent on average in Abi1-deficient clones (Fig. 4A). Abi1 was highly enriched in dorsal ruffles and localized at the tips of ruffles and in the cell periphery in Abi1 control cells but could not be detected in the Abi1-KO cell lines (Fig. 4A). Evaluation of the number and topology of dorsal ruffles revealed an ∼50% decrease in the formation (Fig. 4 B and C) and lower apparent prominence of these structures in Abi1-KO cells (Fig. 4D). This observation indicates an important role of Abi1 in the formation of dorsal ruffles. However, despite the reported critical role of the WAVE2 complex in peripheral ruffle formation (15, 36) and the observed decrease in WAVE2 complex levels, PDGF-induced peripheral ruffling was not affected in Abi1-KO cells (Fig. 4 B and C). Moreover, neither the expression levels of Eps8 and Sos1 nor total Rac activation levels in response to PDGF correlated with the loss of Abi1, indicating that defective Rac activation downstream of the Eps8/Abi1/Sos1 complex does not cause the observed dorsal ruffling phenotype (Fig. S4 B and C).

Fig. 4.

Cell morphologies of Abi1-KO cell lines. (A and B) Localization of Abi1 and WAVE2 in control and Abi1-deficient MEF cells after PDGF stimulation. Control Abi1 (fl/fl) and Abi1 (del/del) cell lines were grown on glass coverslips, serum-starved, and treated with PDGF. Cells then were immunostained with antibodies to Abi1 [anti-Abi1 (4E2)], WAVE2 (#1735), and phalloidin as described in SI Materials and Methods. Abi1 and WAVE2 localized to circular dorsal and peripheral ruffles of precursor cells but were absent (Abi1) or weaker (WAVE2) in these structures in KO cells. (C) Quantification of dorsal and peripheral ruffling. (Left) MEF control (fl/fl), and Abi1-KO cells (del/del) were plated on glass coverslips and serum-starved overnight. Cells were stimulated with PDGF, fixed, and stained with Alexa Fluor 594-conjugated phalloidin to detect F-actin. (Right) The percentages of cells with different cell morphologies in response to PDGF treatment were quantified in the indicated MEF cell lines. Note the reduction of cells displaying dorsal (Upper) but not peripheral (Lower) ruffles in independent Abi1-null clones (c1, c2, d1, d2) compared with their respective control Abi1 (fl/fl) cells (#3 or #8), as indicated. Categories of cell morphologies for quantification were as follows: with ruffles, without ruffles, or with ambiguous morphology. At least 100 cells were analyzed and categorized for each condition. Columns show the percentage of cells of the respective morphology displayed as arithmetic means ± SEM of at least three independent experiments. (D) F-actin staining in PDGF-induced dorsal ruffles in control Abi1 (fl/fl) cells and in Abi1-KO MEF cells [Abi1 (del/del)]. F-actin staining of ruffles revealed different topology and apparent reduction of F-actin in dorsal ruffles in Abi1-KO cells vs. the control cell line. Images were taken from two focal planes, A (Left) and B (Right).

Cells Lacking Abi1 Exhibit Cell-Motility Defects.

Abi1 has been implicated as a regulator of actin cytoskeleton-dependent cell motility, mainly as an integral component of the WAVE2 complex. Therefore, we examined Abi1-KO MEF cell lines in a series of motility assays (Fig. 5). Random cell motility was recorded by video microscopy and was analyzed using the cell-tracking tool (SI Materials and Methods). Control cells migrated an average of 390 μm, whereas KO cells migrated an average of 260 μm. The migration rate of control cells was 0.54 μm/min, compared with 0.36 μm/min for KO cells. However, Abi1-KO cells exhibited increased directional persistence. The net distance vs. true distance ratio was 0.25 for control cells vs. 0.37 for KO cells (arithmetic means, P < 0.0001 for all comparisons). Thus, migration rate and distance were reduced, but directional persistence was increased in Abi1-KO MEF cells compared with control cells. In the wound-healing assay, a statistically significant impairment of wound closure by cells lacking Abi1 was observed: 3 μm²/min by control cells vs. 2.39 μm²/min by KO cells (Fig. 6 and Movies S1, S2, and S3). Taken together, these results indicate that Abi1 removal caused reduced wound-closure rates, probably because of the impairment in cell migration.

Fig. 5.

Impaired motility of MEFs lacking Abi1. The parameters of random cell motility, migration distance, rate, and directional persistence were evaluated in Abi1-null cell lines (c1 and c2) and Abi1 floxed (fl/fl) cells. Cells were seeded under the same conditions and monitored by time-lapse microscopy. Different parameters of cell motility, as indicated above graphs (n = 40 movies per parameter/cell line) were evaluated using ImageJ. Directional persistence was determined as the ratio of total distance traveled over total length of the migration path. Data are presented as ± SEM, based on four independent experiments.

Fig. 6.

Wound-healing migration assay. Cells were seeded under identical conditions. After formation of a confluent monolayer, a wound was produced by scratching with a fine pipette tip. Rate of wound closure was determined by measuring the area remaining uncovered by cells per unit of time. Data represent means ± SEM of four independent assays of Abi1-KO cell lines (c1 and c2) and control (fl/fl) MEF cells.

Discussion

Here we report the production and initial phenotypic characterization of MEF cells lacking a functional Abi1 gene. Among other strategies used to generate Abi1-KO animals and cell lines, targeting of exon 1 has proven successful. Following Cre-mediated recombination, MEF cells lacking Abi1 mRNA and protein expression were obtained. In this study, we focused on the characterization of WAVE2 complex-dependent processes, knowing that participation in the complex is a critical aspect of Abi1 function.

Abi1-KO MEF cells are viable and exhibit no apparent loss of ability to form lamellipodia, stress fibers, or peripheral ruffles. However, Abi1-KO cells displayed decreased efficiency of PDGF-induced dorsal ruffling formation. In addition, dorsal ruffles formed in Abi1-KO cells were much less prominent and displayed less intense F-actin staining compared with control cell lines. Because the levels of the Eps8/Sos1 complex did not correlate with defective dorsal ruffling, we assume that this phenotype is a consequence of the significant instability of WAVE2 complex in the absence of Abi1. The levels of WAVE2 complex components WAVE2, Sra-1/PIR121, and Nap1 were significantly lower in cells lacking Abi1. These data are consistent with previous siRNA knockdown experiments showing that upon down-regulation of individual subunits, the remaining components of the WAVE2 complex are coordinately decreased (11, 37–39). Our results demonstrate that lack of Abi1 caused significant instability of the WAVE2 complex, thus confirming the critical role of Abi1 in the complex function.

In addition to WAVE2, we also observed a decrease in WAVE1 levels, suggesting that Abi1 might be incorporated into complexes with WAVE1, as shown previously (20), and not only with WAVE2. Although WAVE1 originally had been ascribed a role in dorsal but not peripheral ruffle formation (15), this conclusion was contradicted more recently, because WAVE1 KO cells were observed to form multiple dorsal ruffles (36). Likewise, both WAVE1 and WAVE2 previously had been observed to accumulate at the tips of lamellipodia and peripheral ruffles (14, 40), thus indicating that potential functional differences between WAVE1 and WAVE2 are not caused by their differential functions in the formation of peripheral versus dorsal ruffles. Because Abi1-null fibroblasts have reduced expression of both WAVE2 and WAVE1, we speculate that the observed defect in dorsal but not peripheral ruffling simply reflects a higher sensitivity of dorsal ruffles to abrogation of WAVE complex activity rather than differential subcellular functions of WAVE1 versus WAVE2. This hypothesis is consistent with lower levels of total WAVE1/2/3 proteins in Abi1-null fibroblast clones compared with control cells. Consequently, lower WAVE levels cause lower output into Arp2/3 complex activity and F-actin polymerization, as indeed was observed (Fig. 4D). Other consequences of reduced WAVE complex function and Abi1 deficiency are decreased random and directed cellular motility.

Although Abi2 expression was more than doubled in cells lacking Abi1, this response did not compensate for the loss of Abi1 in the apparent loss of integrity of WAVE2 complex, nor did it compensate for loss of Abi1 function pertaining to dorsal ruffling and cellular motility, probably because the amount of Abi2 is too low (demonstrated here to be less than 10% of Abi1 in Abi1-expressing control cells). Nevertheless, the increased amounts of Abi2 and concomitant increase of WAVE3 may well contribute to the relative modesty of phenotypes observed in Abi1-deficient cells.

Abi1 or Abi2 can be incorporated interchangeably into WAVE2 complex. For example, in leukocytes, Abi1 was found in the WAVE2 complex and Abi2 in WAVE1 complex, whereas both Abi1 and Abi2 were present in WAVE2 complex in A431 cells (20). The basis for specificity of Abi protein incorporation into WAVE1 or WAVE2 complex is not known, although, based on high sequence conservation of binding sites, the affinities of the Abi proteins for WAVE proteins are expected to be similar (20, 22, 31). One explanation might be that Abi1 and Abi2 exhibit different patterns of threonine and serine phosphorylation (20), which is required for full activation of WAVE2 complex by Rac and acidic phospholipids (20). Nonetheless, concurrent down-regulation of WAVE1 and WAVE2 in Abi1-KO cells suggests that Abi1 incorporates into both WAVE2 and WAVE1 complexes or, alternatively, that reduced stability of subunits shared by both WAVE1- and WAVE2-containing complexes, such as Sra-1/PIR121 or Nap1, indirectly down-regulates WAVE1 expression. We cannot distinguish between these possibilities at present. Finally, the apparent dysregulation of WAVE3 expression also will have to be explored in the future.

We propose that loss of WAVE2 complex function and dysregulation of WAVE1 and WAVE3 caused by the lack of Abi1 might explain the early embryonic death of Abi1-null embryos. A nonoverlapping function of Abi proteins in embryonic development is indicated by the fact that Abi2 deficiency is not embryonic lethal (27), whereas, as we report here, death of Abi1-null embryos occurred around day E11.5.

The absence of a gene required during developmental progression often is displayed phenotypically by histological abnormalities eventually leading to death at different stages of development. For example, inactivation of murine WAVE2 is lethal by E12.5 (during mid-gestation) (16); inactivation of N-WASP is lethal at E11.0 (during organogenesis) (33, 41); inactivation of Cdc42 is lethal at E3.5 (during implantation) (42); inactivation of Nap1 is lethal at E9.0 (during gastrulation); and inactivation of Rac1 is lethal at E7.5 (during gastrulation) (32, 43, 44). Abi2 mutants are viable and without morphological defects (27). Inactivation mutants of two Sra-1 and HSPC300 genes have not yet been described. Abi1-null embryos developed normally through gastrulation and survived to day E11.5 with marked neural and cardiac defects. Thus, Abi1-signaling events are not required for gastrulation but are critical during brain and heart development. The cardiovascular phenotype is in agreement with observations from a conventional Abi1-KO model (45) that was reported while this manuscript was under consideration.

In summary, we show here that Abi1 loss causes impairments in cellular motility that depend on dynamic actin rearrangements ascribed to WAVE2 activity in actin polymerization. We provide a biochemical explanation for the observed phenotypes, i.e., that loss of Abi1 results in the loss of WAVE2 complex integrity and stability, and that this loss cannot be compensated by the increased, but ultimately insufficient, levels of Abi2. These mechanisms might underlie the critical role of Abi1 in embryonic development. Our conditional Abi1-KO model represents an important tool for addressing the physiological functions of Abi1.

Materials and Methods

Details of methods are presented in SI Materials and Methods. Transgenic ES cells and conditional Abi1-KO mice were generated by conventional methods. MEF lines obtained from conditional Abi-KO mice were used to generate Abi1-null MEF lines by transient transfection with Cre-recombinase. All steps were confirmed by PCR and/or Southern blot. Cell morphologies and marker locations were assessed by confocal and/or epifluorescence microscopy.