ARTICLE Julie Plastino Æ Ioannis Lelidis Æ Jacques Prost Ce´cile Sykes The effect of diffusion, depolymerization and nucleation promoting factors on actin gel growth Received: 30 September 2003 / Accepted: 14 October 2003 / Published online: 9 December 2003 Ó EBSA 2003 Abstract In eukaryotic cells, localized actin polymeriza- tion is able to deform the plasma membrane and push the cell forward. Depolymerization of actin filaments and diffusion of actin monomers ensure the availability of monomers at sites of polymerization, and therefore these processes must play an active role in cellular actin dynamics. Here we reveal experimental evidence that actin gel growth can be limited by monomer diffusion, consistent with theoretical predictions. We study actin gels formed on beads coated with ActA (and ActA fragments), the bacterial factor responsible for actin- based movement of Listeria monocytogenes. We observe a saturation of gel thickness with increasing bead radius, the signature of diffusion control. Data analysis using an elastic model of actin gel growth gives an estimate of 2·10 )8 cm )2 s )1 for the diffusion coefficient of actin monomers through the gel, ten times less than in buffer, and in agreement with literature values in bulk cyto- skeleton, providing corroboration of our model. The depolymerization rate of actin filaments and the elastic modulus of the gel are also evaluated. Furthermore, we qualitatively examine the different actin gels produced when ActA fragments interact with either VASP or the Arp2/3 complex. Keywords Actin polymerization Æ Arp2/3 complex Æ Lamellipodium Æ Motility Æ Vasodilator-stimulated phosphoprotein Introduction The polymerization of actin monomers into filaments against a surface drives the extension of cellular lamellipodia, as well as the intracellular movement of certain vesicles and endosomes, and various pathogens. Much progress has been made in the last five years to- ward understanding the biochemical mechanisms of cellular actin polymerization. Particular emphasis has been placed on the Arp2/3 complex, a component of actin Y-branches (Mullins et al. 1998; Svitkina and Borisy 1999) that is recruited to the leading edge under the control of cellular signaling systems (for review, see Machesky and Insall 1999). The VASP (vasodilator- stimulated phosphoprotein) family proteins also localize to the leading edge of cells, as well as to focal adhesions and stress fibers; however, their role in cell motility is still somewhat controversial (for reviews, see Machesky 2002; Sutherland and Way 2002). Despite the high concentrations of monomeric actin in cells, filaments do not nucleate spontaneously in vivo owing to the presence of the monomer-binding proteins profilin and thymosin-b4. These two proteins work together to maintain a pool of actin ready to polymerize upon the creation of barbed ends (for review, see Pollard et al. 2000). Owing to the abundance of monomeric actin, diffusion does not seem to limit the polymeriza- tion process, although direct experimental evidence is lacking. In fact, the possibility of limitation by diffusion has been suggested theoretically (Noireaux et al. 2000; Mogilner and Edelstein-Keshet 2002), and a recent FLAP (fluorescence localization after photobleaching) study found that diffusion was not sufficient to explain actin delivery to protruding zones in cells (Zicha et al. 2003). We show here the first experimental evidence that, under certain conditions, diffusion of actin monomers is a limiting factor in the growth of an actin gel. For our study, we took advantage of the well-characterized Listeria monocytogenes system, a bacterium that assembles actin filaments at one pole, thereby pushing itself forward within the infected cell. The same proteins as those implicated in leading edge dynamics are involved in this movement: the bacterially produced ActA protein activates actin polymerization nucleation Eur Biophys J (2004) 33: 310–320 DOI 10.1007/s00249-003-0370-3 J. Plastino Æ I. Lelidis Æ J. Prost Æ C. Sykes (&) Laboratoire Physicochimie ‘‘Curie’’, UMR168 Institut Curie/CNRS, 11 rue Pierre et Marie Curie, 75231 Paris cedex 05, France E-mail: cecile.sykes@curie.fr Fax: +33-1-40510636