Contribution of Ena/VASP proteins to intracellular motility of listeria requires phosphorylation and proline-rich core but not F-actin binding or multimerization

Abstract

The Listeria model system has been essential for the identification and characterization of key regulators of the actin cytoskeleton such as the Arp2/3 complex and Ena/vasodilator-stimulated phosphoprotein (VASP) proteins. Although the role of Ena/VASP proteins in Listeria motility has been extensively studied, little is known about the contributions of their domains and phosphorylation state to bacterial motility. To address these issues, we have generated a panel of Ena/VASP mutants and, upon expression in Ena/VASP-deficient cells, evaluated their contribution to Ena/VASP function in Listeria motility. The proline-rich region, the putative G-actin binding site, and the Ser/Thr phosphorylation of Ena/VASP proteins are all required for efficient Listeria motility. Surprisingly, the interaction of Ena/VASP proteins with F-actin and their potential ability to form multimers are both dispensable for their involvement in this process. Our data suggest that Ena/VASP proteins contribute to Listeria motility by regulating both the nucleation and elongation of actin filaments at the bacterial surface.

Figure 1

Motility of L. monocytogenes is impaired in MV^D7 cells. MV^D7 fibroblasts (A and B) and G7 mouse fibroblasts (C and D) were infected with wild-type Listeria (A and C) or the Listeria mutant Δ5 (B and D), fixed, and stained with antibodies against the bacteria (red) and fluorescent phalloidin (green). In G7 mouse fibroblasts wild-type Listeria induced the formation of long actin tails (arrows in C). In contrast, the actin tails induced by these bacteria in MV^D7 cells were much shorter (arrows in A) and closely resembled those induced by the Listeria mutant Δ5 in both cell types (arrows in B and D). Bar, 2 μm.

Figure 2

(A) FACS analysis of MV^D7 cells expressing low (yellow line), medium (red line), and high (light brown line) levels of GFP-tagged Mena and VASP constructs. The black-filled area indicates the background fluorescence of untransfected MV^D7 cells. (B–C′) The expression of wild-type GFP-Mena and GFP-VASP rescues Listeria motility in MV^D7 cells in a concentration-dependent manner. MV^D7 cells expressing high levels of wild-type GFP-Mena or GFP-VASP were infected with Listeria, fixed, and stained with fluorescent phalloidin. In these cells, Listeria recruited GFP-Mena and GFP-VASP at their surface (arrowheads in B′ and C′) and induced the formation of normal actin tails (arrows in B and C; compare with Figure 1). (D) Box and whiskers plots of bacterial speed. Dot indicates the mean, line in the middle of the box indicates the median, top of the box indicates the 75th quartile, whereas the bottom of the box indicates the 25th quartile, and whiskers indicates the 10th and 90th percentiles, respectively. (E–E′) GFP-tagged wild-type Mena and VASP localize at the actin tails induced by the Listeria mutant Δ5. MV^D7 cells expressing high levels of wild-type GFP-Mena were infected with Listeria Δ5, fixed, and stained with fluorescent phalloidin. This Listeria mutant typically induced the formation of short actin tails (arrowhead in E), which were robustly stained with GFP-Mena (arrowhead in E′). Bar (for B–C′ and E–E′), 2 μm.

Figure 3

(A) Schematic diagram of Mena and VASP constructs. Deletions (thick lines) and phosphorylation sites are shown at the bottom and top of Mena and VASP cartoons, respectively. EVH1, Ena-VASP homology 1; PRR, proline-rich region; QRR, glutamine-rich region; EVH2, Ena-VASP homology 2. (B) Western blot analysis of GFP-tagged Mena and VASP fusion proteins. Cell lysates of MV^D7 cells expressing high levels of all Mena, and VASP constructs were resolved by SDS-PAGE, blotted, and probed with an anti-GFP monoclonal antibody. Numbers on the left side of each blot represent molecular weight markers in kilodaltons. (C) Comparison between the expression levels of GFP-tagged wild-type Mena and VASP and their mutated fusion proteins. MV^D7 cells expressing high levels of wild-type GFP-Mena and GFP-VASP (black-filled areas) were compared with MV^D7 cells expressing high levels of each of the GFP-tagged Mena and VASP constructs (red lines) by FACS. The overlapping between the black-filled areas and the red lines in each FACS scan indicates that all cellular populations are equivalent with respect to the expression levels of all Ena/VASP constructs.

Figure 4

Proline-rich region of Ena/VASP proteins is essential for efficient Listeria motility. (A–A") MV^D7 cells expressing high levels of GFP-Mena ΔPRR, GFP-VASP ΔGP₅, or GFP-VASP ΔPRR were infected with wild-type Listeria, fixed, and stained with fluorescent phalloidin. In cell lines expressing GFP-Mena ΔPRR and GFP-VASP ΔPRR Listeria induced the formation of very short actin tails (arrowheads in A and A′), which were morphologically similar to those induced by this bacterium in the parental MV^D7 cells (compare with Figure 1A), whereas Listeria actin tails were significantly longer in cells expressing GFP-VASP ΔGP₅ (arrowheads in A"). Bar, 2 μm. (A) Box and whiskers plots of bacterial speed. (C–D′) Deletion of the proline-rich region of Mena and VASP inhibits the targeting of profilin to the Listeria surface. MV^D7 cells were transfected with CFP-tagged wild-type VASP or VASP ΔPRR and profilin II-YFP and then infected with wild-type Listeria. In cells expressing CFP-VASP, profilin colocalized with VASP at the surface of motile bacteria (arrowheads in C and C′) but not stationary ones (arrows in C and C′). In contrast, in cells expressing CFP-VASP ΔPRR, profilin was absent from the surface of both stationary (arrow in D′) and motile Listeria (arrowhead in D′). Bar, 1 μm.

Figure 5

(A) Deletion of the thymosin β4-like motif of Mena causes a significant decrease of Listeria motility. MV^D7 cells expressing high levels of GFP-Mena ΔTLM were infected with wild-type Listeria, fixed, and stained with fluorescent phalloidin. In these cells, Listeria induced the formation of short actin tails (arrowheads). Bar, 2 μm. (B) Box and whiskers plots of bacterial speed.

Figure 6

(A and B) Deletion of the F-actin–binding site of Mena and VASP increases Listeria motility. MV^D7 cells expressing high levels of GFP-Mena ΔFAB (A) or GFP-VASP ΔFAB (B) were infected with wild-type Listeria, fixed, and stained with fluorescent phalloidin. In both cases, Listeria induced the formation of actin tails (arrows in A and B) that were not distinguishable from those induced by the same bacterium in MV^D7 cells expressing wild-type Ena/VASP proteins (compare to Figure 2). Bar, 2 μm. (C). Box and whiskers plots of bacterial speed.

Figure 7

(A and B) Potential multimerization of Ena/VASP proteins is not required for Listeria motility. MV^D7 cells expressing high levels of GFP-Mena ΔCo-Co (A) or GFP-VASP ΔCo-Co (B) were infected with wild-type Listeria, fixed, and stained with fluorescent phalloidin. In both cell lines, the bacteria induced the formation of actin tails (arrows in A and B) that were not distinguishable from those induced by the same bacterium in MV^D7 cells expressing wild-type Ena/VASP proteins (compare to Figure 2). Bar, 2 μm. (B) Box and whiskers plots of bacterial speed.

Figure 8

(A and B) Phosphorylation of the serine and threonine residues of Mena (A) and VASP (B) increases Listeria motility. Box and whiskers plots of Listeria speed showing the influence of Mena and VASP phosphorylation state on bacterial motility.