Structural insights into de novo actin polymerization
Introduction
The dynamic remodeling of the actin cytoskeleton is essential for many cellular functions, including cell locomotion, cytokinesis, and membrane trafficking. Additionally, many pathogens hijack the host cell actin cytoskeleton during infection. These processes involve rapid bursts of actin polymerization/depolymerization. But, somewhat paradoxically, the formation of new actin filaments in cells is kinetically unfavorable, because of the instability of polymerization intermediates (actin dimers, trimers, and tetramers) and the actions of actin monomer binding proteins such as profilin and thymosin-β4 (Tβ4). This creates an opportunity for cells to actively regulate the de novo polymerization of actin by using actin filament nucleation and elongation factors.
The actin filament can be described as either a single left-handed short-pitch helix, with consecutive lateral subunits staggered with respect to one another by half a monomer length, or two right-handed long-pitch helices of head-to-tail bound actin subunits [1]. Filament nucleators work by different mechanisms, stabilizing small actin oligomers along either the long-pitch helices or the short-pitch helix of the actin filament. With the exception of formins they all use the WASP-Homology 2 (WH2 or W) domain, a small and versatile actin-binding motif, for interaction with actin. In various proteins, including Spire, Cobl, and VopL/VopF, the W domain occurs in tandem repeats that bind three to four actin subunits to form a nucleus. Structural considerations suggest that Nucleation Promoting Factors (NPFs), proteins that form a nucleus consisting of one to three actin subunits and the two actin-related proteins of Arp2/3 complex, can be viewed as a specialized form of tandem W domain nucleator. Formins are unique in that they use the formin-homology 2 (FH2) domain for interaction with actin and promote not only nucleation, but also processive barbed end elongation. By contrast, the elongation function among W-based nucleators has been outsourced to a dedicated family of proteins, Eva/VASP, which are related to NPFs.
Section snippets
Formins
Among filament nucleation/elongation factors, formins are the best understood at the structural level [2, 3, 4]. In cellular processes ranging from cell locomotion and morphogenesis to cytokinesis, formins mediate the assembly of unbranched actin networks, such as filopodia, stress fibers, and actin cables. Like most cytoskeletal proteins, formins are multidomain, multifunctional proteins. There is significant sequence variability within the formin family, but two regions of higher conservation
Tandem W domain-based filament nucleators
With the exception of formins, filament nucleators use the W domain for interaction with actin, although in Lmod and Arp2/3 complex other domains contribute as well. The W domain is short (17–27-aa) and poorly conserved [13]. Its N-terminal portion forms a helix that binds in the target-binding cleft between actin subdomains 1 and 3 [14, 15••] (Figure 2). The extended region after this helix has variable length and sequence, but comprises the conserved motif LKKT(V).
The W domain often occurs in
Filament nucleation by NPFs–Arp2/3 complex
The W domain also participates in filament nucleation through NPFs of the Arp2/3 complex, which have between one and three W domains (Figure 4a). Arp2/3 complex consists of seven proteins, including two actin-related proteins, Arp2 and Arp3, and subunits ARPC1–5 (Figure 4b). By itself, Arp2/3 complex has low nucleation activity [21]. The discovery of ActA as a NPF at the surface of Listeria monocytogenes [22] and eukaryotic NPFs of the WASP/WAVE family [23, 24, 25, 26] revealed the strong
Ena/VASP proteins as dedicated elongation factors among W-based filament nucleators
While formins promote both nucleation and elongation and use the FH2 domain for both activities, none of the W-based nucleators seems to promote elongation. This function appears to have been outsourced to a dedicated family of W-containing proteins, Ena/VASP, which in turn play no role on nucleation [47]. The domain organization of Ena/VASP is somewhat similar to that of WASP/N-WASP. Both contain N-terminal EVH1 (or WH1) domains, central Pro-rich regions and W-related sequences, known as the
Conclusions
The structural biology of actin filament nucleation and elongation has been a hot topic of research in recent years, owing to the pivotal role these processes play in numerous cellular functions. The proteins involved are numerous, work by different mechanisms, and function under the control of diverse regulatory pathways. Significant progress has been made in our understanding of formin structure–function. The determination of atomic structures of Arp2/3 complex, W-actin complexes, and
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
Supported by NIH grants GM073791.
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