Actin pedestal formation by enteropathogenic Escherichia coli and intracellular motility of Shigella flexneri are abolished in N-WASP-defective cells

Abstract

In mammalian cells, actin dynamics is tightly controlled through small GTPases of the Rho family, WASP/Scar proteins and the Arp2/3 complex. We employed Cre/loxP-mediated gene targeting to disrupt the ubiquitously expressed N-WASP in the mouse germline, which led to embryonic lethality. To elucidate the role of N-WASP at the cellular level, we immortalized embryonic fibroblasts and selected various N-WASP-defective cell lines. These fibroblasts showed no apparent morphological alterations and were highly responsive to the induction of filopodia, but failed to support the motility of Shigella flexneri. In addition, enteropathogenic Escherichia coli were incapable of inducing the formation of actin pedestals in N-WASP-defective cells. Our results prove the essential role of this protein for actin cytoskeletal changes induced by these bacterial pathogens in vivo and in addition show for the first time that N-WASP is dispensable for filopodia formation.

Fig. 1. Disruption of N-WASP leads to embryonic lethality. (A) Schematic representation of the N-WASP protein and the gene targeting strategy showing the genomic organization of the N-WASP locus in wild-type, targeted, floxed and deleted alleles. Also shown are the targeting vector and external and internal probes used for Southern blot analysis. Note the introduction of a new EcoRV restriction site upon gene targeting (marked EcoRV*) used to distinguish the different alleles. (B and C) Southern analyses of precursor (wt), targeted (a and b), floxed (N-WASP^flox/wt) (a′, b′), and deleted (N-WASP^del/wt) (b″) ES clones. (D) Southern analysis of N-WASP^flox/wt mice (marked with asterisks) derived after germline transmission. (E) PCR for genotypes of mice (lanes 1–4) and fibroblast cell lines (i and ii): wild-type, floxed and deleted alleles. Note the absence of the N-WASP^flox allele in the N-WASP^del/del cell line (ii). (F) Western blot analysis of N-WASP in extracts of hippocampi of wild-type (c), N-WASP^flox/flox (d) and N-WASP^flox/flox Cre159 mice (e) with polyclonal antibodies against the N-terminus of N-WASP. (G) Southern and (H) Western blot analyses of a representative N-WASP^flox/flox fibroblast precursor cell line (i) and its corresponding N-WASP^del/del line (ii). (H) Note the lack of N-WASP full-length expression in (ii). In both (F) and (H), the crossreaction at ∼30 kDa was used as loading control. The small polypeptide of ∼23 kD detected in extracts of N-WASP^del/del fibroblasts is indicated by an arrow. This product was not detected in forebrain extracts from Cre159 transgenic animals (F, lane e). (I) Comparison of genotyped embryos from intercrosses of N-WASP^del/wt mice at E11.5. Note the reduced size and developmental retardation of the N-WASP^del/del embryo (right) as compared with its wild-type sibling (left), H points to heart. Bars, 1 mm. (J) Histological analyses of presumptive wild-type (left) and N-WASP^del/del embryo (right) at E9 from intercrosses of N-WASP^del/wt mice. The cranial parts of the wild-type embryo are well developed showing pharyngeal arches, a large brain cavity and an almost closed cranial neuropore in the hindbrain area (filled arrowhead). The heavily retarded embryo has not turned, but has formed a heart (marked H) and cranial mesenchyme, whereas the allantois (short arrow) shows widening of the intercellular spaces distally and does not contribute to the placenta as in wild-type. Open arrowheads point at amnion. Bars, 150 µm.

Fig. 1. Disruption of N-WASP leads to embryonic lethality. (A) Schematic representation of the N-WASP protein and the gene targeting strategy showing the genomic organization of the N-WASP locus in wild-type, targeted, floxed and deleted alleles. Also shown are the targeting vector and external and internal probes used for Southern blot analysis. Note the introduction of a new EcoRV restriction site upon gene targeting (marked EcoRV*) used to distinguish the different alleles. (B and C) Southern analyses of precursor (wt), targeted (a and b), floxed (N-WASP^flox/wt) (a′, b′), and deleted (N-WASP^del/wt) (b″) ES clones. (D) Southern analysis of N-WASP^flox/wt mice (marked with asterisks) derived after germline transmission. (E) PCR for genotypes of mice (lanes 1–4) and fibroblast cell lines (i and ii): wild-type, floxed and deleted alleles. Note the absence of the N-WASP^flox allele in the N-WASP^del/del cell line (ii). (F) Western blot analysis of N-WASP in extracts of hippocampi of wild-type (c), N-WASP^flox/flox (d) and N-WASP^flox/flox Cre159 mice (e) with polyclonal antibodies against the N-terminus of N-WASP. (G) Southern and (H) Western blot analyses of a representative N-WASP^flox/flox fibroblast precursor cell line (i) and its corresponding N-WASP^del/del line (ii). (H) Note the lack of N-WASP full-length expression in (ii). In both (F) and (H), the crossreaction at ∼30 kDa was used as loading control. The small polypeptide of ∼23 kD detected in extracts of N-WASP^del/del fibroblasts is indicated by an arrow. This product was not detected in forebrain extracts from Cre159 transgenic animals (F, lane e). (I) Comparison of genotyped embryos from intercrosses of N-WASP^del/wt mice at E11.5. Note the reduced size and developmental retardation of the N-WASP^del/del embryo (right) as compared with its wild-type sibling (left), H points to heart. Bars, 1 mm. (J) Histological analyses of presumptive wild-type (left) and N-WASP^del/del embryo (right) at E9 from intercrosses of N-WASP^del/wt mice. The cranial parts of the wild-type embryo are well developed showing pharyngeal arches, a large brain cavity and an almost closed cranial neuropore in the hindbrain area (filled arrowhead). The heavily retarded embryo has not turned, but has formed a heart (marked H) and cranial mesenchyme, whereas the allantois (short arrow) shows widening of the intercellular spaces distally and does not contribute to the placenta as in wild-type. Open arrowheads point at amnion. Bars, 150 µm.

Fig. 2. N-WASP is not required for Cdc42-induced filopodia formation. Cells were microinjected with a mixture of L61Cdc42 (1.5mg/ml), N17Rac (0.35 mg/ml) and C3-transferase (0.1 mg/ml). Phase contrast images show a precursor (A, A′) and a corresponding N-WASP-defective cell (B, B′) before (A, B) and after (A′, B′) microinjection, which induced the formation of filopodia in both cell types. Approximate times before and after injections are given in minutes and seconds. Bar, 5 µm.

Fig. 3. N-WASP is essential for Shigella-induced actin tail formation. Precursor (B) and N-WASP-defective fibroblasts (A and C–J) were infected with L. monocytogenes (A) or S. flexneri (B–J). (A–C) show non-transfected cells, while (D–J) show cells expressing GFP-tagged N-WASP constructs as indicated. In all images, filamentous actin is shown in red. Listeria (A) and Shigella (B, C) are shown in green. In (D–J), Shigella and GFP fusion proteins are labeled blue and green, respectively. Listeria form actin tails in N-WASP-defective fibroblasts (A), while Shigella-induced actin tails are abolished in these cells (C), in contrast to the precursor cell line (B). Actin tail formation by Shigella is restored upon expression of full-length N-WASP (D), H208D, Δ(B-CRIB) and Δ(WH1-CRIB) mutants (E, F and J, respectively), but not after expression of N-WASP-WH1 (G), -B-GBD (H) or N-WASP-ΔWA (I), although the three latter constructs are recruited to the Shigella surface (indicated by arrows). Bar in (A) (5 µm) is valid for (A–J) except (C) (10 µm). (K) shows the domain structure of N-WASP and an overview of the GFP-tagged constructs used in this study. Recruitment to Shigella and reconstitution of actin tail formation are given. +* marks low efficiency and/or shorter tails; PH, pleckstrin-homology domain; IQ, calmodulin-binding motif; B, basic domain; GBD, GTPase binding domain; polyPro, polyproline region; V, verprolin homology domain; C, cofilin homology domain and A, acidic domain.

Fig. 4. EPEC pedestal formation depends on N-WASP. Infection of precursor (A, B) and N-WASP-defective cells (C–J) with EPEC. EPEC are shown in (A, C and H) and in blue in (E–G). F-actin is shown in (B, D and J) and in red in (E–G). GFP constructs are shown in (I) and in green in (E–G). EPEC (A, C)-induced formation of actin pedestals in precursor (B), but not in N-WASP-defective cells (D). Pedestal formation in N-WASP-defective cells was restored upon expression of GFP-tagged full-length N-WASP (E), N-WASP-H208D (F) and N-WASP-Δ(B-CRIB) (G), while N-WASP-WH1-GBD was recruited (I) by EPEC (H) without induction of actin pedestals (J). Bars (5 µm) in (A, E and H) are valid for (A–D), (E–G) and (H–J), respectively. For description of GFP fusion constructs see Figure 3K. (K) The recruitment to EPEC attachment sites and reconstitution of pedestal formation by the respective mutants are indicated.