Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, alpha-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.

WAVE2 belongs to a family of proteins that mediates actin reorganization by relaying signals from Rac to the Arp2/3 complex, resulting in lamellipodia protrusion. WAVE2 displays Arp2/3-dependent actin nucleation activity in vitro, and does not bind directly to Rac. Instead, it forms macromolecular complexes that have been reported to exert both positive and negative modes of regulation. How these complexes are assembled, localized and activated in vivo remains to be established. Here we use tandem mass spectrometry to identify an Abi1-based complex containing WAVE2, Nap1 (Nck-associated protein) and PIR121. Abi1 interacts directly with the WHD domain of WAVE2, increases WAVE2 actin polymerization activity and mediates the assembly of a WAVE2-Abi1-Nap1-PIR121 complex. The WAVE2-Abi1-Nap1-PIR121 complex is as active as the WAVE2-Abi1 sub-complex in stimulating Arp2/3, and after Rac activation it is re-localized to the leading edge of ruffles in vivo. Consistently, inhibition of Abi1 by RNA interference (RNAi) abrogates Rac-dependent lamellipodia protrusion. Thus, Abi1 orchestrates the proper assembly of the WAVE2 complex and mediates its activation at the leading edge in vivo.

Single actin filaments undergoing brownian movement in two dimensions were observed at 20 degrees C in fluorescence optical video microscopy. The persistence length (Lp) was derived from the analysis of either the cosine correlation function or the average transverse fluctuations of a series of recorded shapes of filaments assembled from rhodamine-action. Phalloidin-stabilized filaments had a persistence length of 18 +/- 1 micron, in agreement with recent observations. In the absence of phalloidin, rhodamine-labeled filaments could be observed under a variety of solution conditions once diluted in free unlabeled G-actin at the appropriate critical concentration. Such nonstabilized F-ADP-actin filaments had the same Lp of 9 +/- 0.5 microns, whether they had been assembled from ATP-G-actin or from ADP-G-actin, and independently of the tightly bound divalent metal ion. In the presence of BeF3-, which mimics the gamma-phosphate of ATP, F-ADP-BeF3-actin was appreciably more rigid, with Lp = 13.5 microns. Hence, newly formed F-ADP-Pi-actin filaments are more rigid than "old" F-ADP-actin filaments, a fact which has implications in actin-based motility processes. In the presence of skeletal tropomyosin and troponin, filaments were rigid (Lp = 20 +/- 1 micron) in the off state (-Ca2+), and flexible (Lp = 12 microns) in the on state (+Ca2+), consistent with the steric blocking model. In agreement with x-ray diffraction data, no appreciable difference was recorded between the off and on states using smooth muscle tropomyosin and caldesmon (Lp = 20 +/- 1 micron). In conclusion, this method allows accurate measurement of small (< or = 15%) changes in mechanical properties of actin filaments in correlation with their biological functions.

Intracellular propulsion of Listeria monocytogenes is the best understood form of motility dependent on actin polymerization. We have used in vitro motility assays of Listeria in platelet and brain extracts to elucidate the function of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility. Immunodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like protein) from brain extracts of Mena knockout (−/−) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena, and Evl play interchangeable roles and are required to transform actin polymerization into active movement and propulsive force. The EVH1 (Ena/VASP homology 1) domain of VASP is in slow association–dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA. VASP also interacts with F-actin via its COOH-terminal EVH2 domain. Hence VASP/ Ena/Evl link the bacterium to the actin tail, which is required for movement. The affinity of VASP for F-actin is controlled by phosphorylation of serine 157 by cAMP-dependent protein kinase. Phospho-VASP binds with high affinity (0.5 × 108 M−1); dephospho-VASP binds 40-fold less tightly. We propose a molecular ratchet model for insertional polymerization of actin, within which frequent attachment–detachment of VASP to F-actin allows its sliding along the growing filament.