The in vitro motility assay of L
in platelet and brain extracts has been used as a tool to demonstrate that proteins of the Ena/VASP family are required for actin-based motility. Since active actin polymerization, but no movement, is observed around the bacteria after selective removal of VASP from platelet extracts (or of Mena and Evl from brain extracts), we conclude that these proteins do not act as actual nucleators of actin assembly, but as organizers of the actin meshwork for the efficient production of propulsive force. This conclusion is in full agreement with the one derived from genetic studies showing that the deletion of the four proline-rich repeats of ActA caused a severe inhibition of Listeria
movement in infected cells and in Xenopus
egg extracts (Smith et al., 1996
). Although bacteria expressing modified ActA still induced actin assembly, the number of bacteria moving (albeit at a fourfold reduced speed) was 10-fold lower than for wild-type bacteria. A 5.5-fold reduced speed of movement in infected cells was also observed in a second report (Niebuhr et al., 1997
). The quantitative difference between the observations derived from genetically modified Listeria
and the present in vitro VASP/Mena/Evl depletion studies lies in the fact that in the previous experiments, proteins of the Ena/VASP family were still present in the medium. Therefore, the possibility cannot be discounted that low-affinity binding of these proteins to subsites on ActA, undetectable by immunofluorescence, would be sufficient to elicit slow movement of a few bacteria. Interestingly, a motility phenotype similar to the one obtained upon VASP removal (actin polymerization around the bacteria, indicating that Arp2/3 was recruited, but no actin tail formation and no movement) was observed upon deletion of the 117-KKRRK-121 peptide in the NH2
-terminal region of ActA (Lasa et al., 1997
). Although VASP was bound to these NH2
-terminal deletion ActA mutants, the phenotype suggests that it may not have been bound in a functional fashion. In summary, while the proline repeats of ActA may represent the main VASP binding subsite, other regions of ActA may also be involved in VASP function.
VASP, Mena, and Evl appear to play interchangeable roles in actin-based motility, in different cellular contexts. Evl can replace VASP in VASP-depleted platelet extracts, and VASP can replace both Mena and Evl in Mena knockout, Evl-depleted brain extracts. This conclusion is in agreement with a recent report (Ahern-Djamali et al., 1998
) showing that, in vivo, human VASP can substitute for the loss of Ena in the Drosophila
embryo. It shows that these proteins are likely to interact with the same targets in different cells, and that the role of Ena in neural development may be, in analogy with the function of VASP in Listeria
movement, to organize the growth of filaments at the plasma membrane for the correct development of neural cells. Whether VASP, Ena, Mena, and Evl are similarly regulated in different tissues is an open question.
Ena/VASP proteins clearly can support Listeria
movement independently of profilin, since the simple add-back of VASP to extracts that have been double-depleted from VASP and profilin is sufficient to restore movement of Listeria
at a rate which is 75% of the rate observed upon add-back of both VASP and profilin. Listeria
movement does not require profilin, however the increase in bacterial speed provided by profilin is consistent with its function in filament turnover (Didry et al., 1998
). In the presence of ADF (2 μM in platelet extracts), profilin increases the turnover of actin filaments, which powers the movement of Listeria
. Whether an additional effect is provided by the interaction of VASP/Mena with profilin is an issue which is not elucidated by our work. The engineering of mutated forms of VASP unable to bind profilin but binding ActA and F-actin in an unaltered fashion, or of mutated forms of profilin unable to bind poly-l
-proline but interacting with actin only, in a complex able to participate in barbed end assembly like unmodified profilin appears required to clarify this point. In this context, it is noteworthy that a recent report (Suetsugu et al., 1998
) studying the role of profilin in N-WASP–elicited filopodia extension in response to EGF demonstrates that the ability of profilin to interact directly with actin is required for N-WASP–mediated filopodial extension, and that mutations in the proline-rich region of N-WASP which abolish the binding of profilin to N-WASP did not impair filopodium extension, although the length of the microspikes was greatly reduced.
The in vitro motility assay of Listeria provides quantitative estimates of the affinities of the different domains of the Ena/VASP proteins involved in building the molecular scaffold responsible for actin assembly and movement. The recombinant VASP and Evl proteins used in this work were efficient at submicromolar concentrations. Use of the GST-EVH1 and GST-(Pro-ActA) fusion proteins of VASP, Mena, and Evl demonstrates that the EVH1 domain itself binds the ActA proline-rich region in slow association–dissociation equilibrium, and with an affinity which, given the very slow dissociation rate, we tentatively estimate at least in the 107 M−1 range. This implies that the attachment of VASP to ActA is quasi-permanent during the movement of Listeria.
VASP interacts also with actin via its EVH2 domain. The isolated EVH2 domain binds F-actin in a 1:1 molar ratio and an affinity of 0.7 μM, identical to the binding stoichiometry and affinity of dephospho-VASP for F-actin. The fact that EVH2, in displacing VASP from F-actin, abolishes the movement, demonstrates that the interaction of VASP with the filaments of the actin tail is essential in propulsion. In binding its two targets, ActA and actin, VASP may work as a molecular connector linking the surface of the bacterium to the growing filament via its EVH1 and EVH2 domains. This result is in full agreement with the strong bonding between the bacterium and the actin tail measured with optical tweezers (Gerbal, F.B., V. Laurent, A. Ott, P. Chaikin, M.-F. Carlier, and J. Prost, manuscript in preparation). Our results expand the conclusion of these mechanical studies in showing that the attachment of the actin tail to the bacterium is required for movement, a conclusion at variance with the original Brownian ratchet model (Peskin et al., 1993
). One can imagine that the connection elicited by VASP between ActA and actin imposes a structural constraint in the arrangement of the elongating barbed ends at the surface of Listeria
, maintaining them attached to the bacterium and bundled in a defined orientation, which would allow the development of force in privileged location and direction. In the absence of VASP, the growing filament ends are randomly oriented, leading to the formation of disorganized actin clouds from which a productive force against the bacterium wall cannot result. The EVH2 domain of Ena has been proposed recently to be involved in multimerization of Ena (Ahern-Djamali et al., 1998
). In view of the present data showing the interaction of EVH2 with actin, it is also possible that F-actin was present as a third partner serving to mediate or stabilize the interaction. More detailed investigations will have to be carried out to address this possibility.
The present data lead us to formulate the following model. During Listeria
movement, VASP remains located at the bacterium–actin tail interface, implying that while the EVH1 domain is bound to ActA, the EVH2 domain slides along the side of the barbed end of the growing filament, allowing insertional polymerization of actin. This working model of a molecular ratchet is illustrated in Fig. . Within this model, in maintaining the site of filament growth at a defined location and distance from the bacterium surface, each added subunit would contribute in pushing the bacterium, hence VASP would increase the yield of the transformation of actin polymerization into propulsive force. Since statistically all filaments do not attach and detach simultaneously, considering the collective behavior of the population of filaments assembled at the bacterium surface is essential in understanding the mechanism of movement. Factors controlling the strength of VASP interaction with actin, i.e., the frequency of attachment–detachment steps, are expected to regulate the filament sliding rate, hence to control the speed of propulsion of Listeria
. The fact that the affinity of VASP for F-actin is increased 40-fold (up to 0.5 × 108
) by phosphorylation of serine 157 raises the possibility that cAMP-dependent phosphorylation of VASP slows down the movement of Listeria
. This possibility is currently under investigation. Our data and conclusions compare well with those derived from a recent study (Aszodi et al., 1999
) showing that VASP-null platelets aggregate faster. This observation suggests that the role of VASP, which would be enhanced by phosphorylation, could be in part, via actin binding, to maintain the cohesive integrity of the membrane actin cytoskeleton of unstimulated platelets. In conclusion, the use of selectively depleted platelet and brain extracts provides a useful tool for elucidating the role of Ena/VASP family proteins in Listeria
motility and in normal cellular function.
Figure 9 Working model for the role of VASP in actin-based motility. This scheme summarizes the conclusions from the present work concerning the role of VASP in Listeria movement. Step 0 represents nucleation of actin filaments on Arp2/3 complex in interaction (more ...)