https://pmc.ncbi.nlm.nih.gov/articles/PMC3603506/
Mechanism of actin nucleation
The precise mechanism by which formins nucleate the assembly of actin filaments is still being worked out. Initially, it was proposed that formins nucleate actin filaments by capturing and stabilizing spontaneously formed actin dimers and trimers (Pring et al., 2003) (see Poster), on the basis that the FH2 domain alone is sufficient for nucleation in vitro but lacks detectable binding affinity for actin monomers.
Subsequently, however, it was shown that FH2-domain-mediated nucleation is very inefficient when using profilin-bound actin monomers, the predominant substrate that is available for actin polymerization in cells (Chesarone et al., 2010). More recent studies have shown that the C-terminal tail regions of formins bind actin monomers and enhance nucleation in the presence of profilin, which might explain how formins nucleate actin assembly in vivo (Gould et al., 2011; Heimsath and Higgs, 2012).
In addition, the tail regions of formins can interact with other factors that bind actin monomers with high affinity and promote nucleation (see below). Thus, the interplay between formins and nucleation co-factors is a powerful means by which both, tight control and robust stimulation of actin nucleation, can be achieved in vivo (Blanchoin and Michelot, 2012).
Moreover, interactions between FH1 and the profilin-actin complex might contribute to nucleation (Paul and Pollard, 2008). Thus, nucleation triggered through formins in the presence of profilin may involve contributions by their FH1 and FH2 domains, and the tail regions.
Although most formins promote actin nucleation and elongation, the strength of these activities can vary drastically. For example, S. pombe Cdc12 is a potent nucleator with a nucleation efficiency of over 50% (every second formin dimer nucleates a filament) (Neidt et al., 2008), whereas other formins, such as Daam1, FMNL3 (also known as FRL2) and FMNL2</emph> have nucleation efficiencies of below 1% (Vaillant et al., 2008; Block et al., 2012). These differences may reflect diverse in vivo requirements, e.g. the need for a slower and more controlled nucleation in some instances, or the dedication of a formin to actin filament elongation rather than nucleation. Indeed, one study has proposed that the primary role of FMNL2 at the leading edge of the cell is to capture free actin filament barbed ends that are nucleated by the Arp2/3 complex and elongate those filaments to drive filopodial and lamellipodial extension (Block et al., 2012). The weaker nucleation by some formins that has been observed in vitro might also reflect their stronger dependence on co-factors or nucleation promoting factors (NPFs) in vivo.
Formin-interacting NPFs are believed to help formins overcome the barrier that profilin constitutes to actin nucleation. Profilin suppresses the self-association of actin into dimers and trimers, and reduces the efficiency of formin nucleation (Neidt et al., 2008; Paul and Pollard, 2008; Scott et al., 2011). However, NPFs effectively compete with profilin for actin monomer binding, and organize mutiple actin monomers in proximity to the formin FH2 domain. Such NPF-formin pairs include Bud6–Bni1, Spire–FMN (Spir and Cappuccino in Drosophila) and adenomateous polyposis coli protein (APC)–mDia1 (Moseley et al., 2004; Quinlan et al., 2007; Webb et al., 2009; Okada et al., 2010; Graziano et al., 2011; Tu et al., 2012). In vivo, Bni1–Bud6 and APC–mDia1 function together to assemble actin cables and pseudocleavage furrows, respectively, and have also been shown to directly interact in vitro to assemble actin in the presence of profilin and/or capping protein (Moseley et al., 2004; Okada et al., 2010; Graziano et al., 2011; Breitsprecher et al., 2012). Spire and FMN co-function in vivo to assemble cytoplasmic actin meshworks (Schumacher et al., 2004; Pfender et al., 2011; Schuh, 2011) but, perplexingly, Spire inhibits rather than enhances the nucleation activity of FMN in vitro (Quinlan et al., 2007; Vizcarra et al., 2011; Zeth et al., 2011), suggesting that additional factors are required to activate collaborative actin assembly through Spire–FMN. More recently, we have begun to address the question of how NPF–formin pairs co-assemble actin filaments by using triple-color total internal reflection fluorescence (TIRF) microscopy at the single-molecule level (Breitsprecher et al., 2012). This study revealed that, during the early phases of nucleation, mDia1 and APC molecules associate, with APC being mainly responsible for recruiting actin monomers. Subsequently, upon actin polymerization, APC and mDia1 separate, with APC remaining at the nucleation site and mDia1 moving processively along the growing barbed end, where it protects the filament from capping protein (Breitsprecher et al., 2012). An important next goal will be to determine whether other NPF–formin pairs use similar or distinct mechanisms.
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