A large number of cellular processes, including cytokinesis, endocytosis, chemotaxis, or neurite outgrowth, is mediated by polymerization of actin filaments. In response to extracellular stimuli, motile protrusions of the plasma membrane, in the form of lamellipodia or filopodia, are driven by the continuous initiation, polarized growth, and turnover of actin filaments at the leading edge of the cell.Recent progress has been made in identifying the key players responsible for the spatio-temporal control of actin-based motility and in understanding the molecular mechanism supporting their function. The linkage of the actin cytoskeleton to the signaling pathway is generally mediated by the interaction of a small Gprotein (Rac, Cdc42) in its active, GTP-bound form, with a multipartner "connector" at the plasma membrane. The activation of the connector allows it to recruit the Arp2/3 complex, which initiates branched barbed end growth of actin filaments (see the first minireview in this series (60)) forming the dense reticulated actin network seen in high resolution electron microscopy images of lamellipodia (1-3).Pathogens like Listeria or Shigella or the vaccinia virus, which propel themselves through the cytoplasm by polymerizing actin at their surface (4), harness the cytoskeletal machinery downstream of the signaling pathway in a constitutive fashion and provide a biochemical approach of the mechanism of actin-based motility.Once actin polymerization is initiated, continuous filament growth causes membrane protrusion (or bacterium propulsion) at a rate of 1-25 m/min. Growing filaments remain stationary with respect to the substratum (5), demonstrating that actin polymerization is linked to the movement. Barbed end growth of filaments initiated at the leading edge (or at the surface of Listeria or Shigella) is fed by subunits provided by continuous depolymerization of filaments from their pointed ends at the rear of the lamellipodial extension, which is thus maintained at a constant width (6). Conversely, the actin tail formed at the rear of Listeria or Shigella remains at a constant length in a stationary regime of propulsion (7). The steady polymerization reflects actual turnover of actin filaments according to a treadmilling process (8). One of the fascinating aspects of actin-based motility is the rapid rate of filament turnover, which supports the movement. Typically, to push the membrane forward at 10 m/min, individual barbed ends must readily incorporate 100 subunits/s. This rate is 200-fold higher than the treadmilling rate measured in vitro for pure F-actin turnover at steady state. From a thermodynamic point of view, both the activated nucleation and the rapid barbed end growth of filaments imply that upon cell stimulation, the concentration of monomeric ATP-actin is increased above its level in quiescent cells, so as to promote nucleation, and remains high, well above the critical concentration for barbed end assembly during movement, so as to sustain rapid steady filament growth. Recent works have sh...

The thermodynamics and kinetics of actin interaction with Arabidopsis thaliana actin-depolymerizing factor (ADF) 1 , human ADF, and S6D mutant ADF 1 protein mimicking phosphorylated (inactive) ADF are examined comparatively. ADFs interact with ADP⅐G-actin in rapid equilibrium (k ؉ ‫؍‬ 155 M ؊1 ⅐s ؊1 and k ؊ ‫؍‬ 16 s ؊1 at 4°C under physiological ionic conditions). The kinetics of interaction of plant and human ADFs with F-actin are slower and exhibit kinetic cooperativity, consistent with a scheme in which the initial binding of ADF to two adjacent subunits of the filament nucleates a structural change that propagates along the filament, allowing faster binding of ADF in a "zipper" mode. ADF binds in a non-cooperative faster process to gelsolin-capped filaments or to subtilisin-cleaved F-actin, which are structurally different from standard filaments (Orlova, A., Prochniewicz, E., and Egelman, E. H. (1995) J. Mol. Biol. 245, 598 -607). In contrast, the binding of phalloidin to F-actin cooperatively inhibits its interaction with ADF. The ADF-facilitated nucleation of ADP⅐actin self-assembly indicates that ADF stabilizes lateral interactions in the filament. Plant and human ADFs cause only partial depolymerization of F-actin at pH 8, consistent with identical functions in enhancing F-actin dynamics. Phosphorylation does not affect ADF activity per se, but decreases its affinity for actin by 20-fold.A large body of evidence supports the view that the rapid turnover of actin filaments drives actin-based motility processes such as the forward movement of the leading edge of the lamellipodium of locomoting cells, the propulsive movement of Listeria monocytogenes, the movement of cortical actin patches in yeast, or the extension of the growth cone (1). Filaments turn over via a treadmilling mechanism, whereby the steady growth of barbed ends is fed by the subunits depolymerizing from the pointed ends (2, 3). While barbed end growth, which provides the motile force (4), is restricted to specialized regions of the cell such as the leading edge (5), depolymerization may occur from all pointed ends in the cell medium. The observed rates of actin-based movement fall in the range 1-20 m/min, corresponding to treadmilling rates of 7-130 subunits/s/filament, i.e. 1-2 orders of magnitude higher than the treadmilling rate measured in vitro in solutions of pure actin (6).Actin-binding proteins of the ADF 1 /cofilin family have recently been demonstrated to enhance the treadmilling of Factin in vitro by 25-fold and consistently to increase the rate of Listeria propulsion in platelet extracts (7). We proposed (7) that these in vitro properties of ADF accounted for the enhancement of motility of Dictyostelium discoideum (8) due to ADF overexpression and for its high level of expression in early development (9). Consistently, ADF was shown to be responsible for the high rate of filament turnover in yeast (10).It was initially thought that ADF depolymerized F-actin rapidly due to a severing activity (11)(12)(13)(14). Severing of the f...

New "molecular tongs" based on naphthalene and quinoline scaffolds linked to two peptidic strands were synthesized. They were designed to prevent dimerization of HIV-1 protease by targeting the antiparallel beta-sheet involving N- and C-termini of each monomer. Compared to "molecular tongs" previously described (Bouras, A.; Boggetto, N.; Benatalah, Z.; de Rosny, E.; Sicsic, S.; Reboux-Ravaud, M. J. Med. Chem. 1999, 42, 957-962), two main different structural features were introduced: positively charged quinoline as a new scaffold and two peptidic strands displaying different sequences. Seventeen new "molecular tongs" with dipeptidic or tripeptidic strands were synthesized. These molecules were assayed on HIV-1 protease using the Zhang kinetic technique. Eleven molecules behaved as pure dimerization inhibitors, mostly at the submicromolar range. Compared to a naphthalene scaffold, the quinoline one was shown in several cases to favor dimerization inhibition. The simplified hydrophobic Val-Leu-Val-OMe strand was confirmed as particularly favorable. The C-terminal analogue strand Thr-Leu-Asn-OMe was shown to be the best one for inducing dimerization inhibition (K(id) of 80 nM for compound 30). The mechanism of inhibition was ascertained using ANS binding and gel filtration. Experimental results are in agreement with the dissociation of the HIV-1 protease dimeric form in the presence of the synthesized molecular tongs.