Actin filaments (F-actin) are linear polymers of globular actin (G-actin) subunits and occur as microfilaments in the cytoskeleton and as thin filaments, which are part of the contractile apparatus, in muscle and nonmuscle cells. They commonly underlie the plasma membrane and are typically assembled at the cell periphery from adhesion sites or sites of membrane extension. Actin filaments can create a number of linear bundles, two-dimensional networks, and three-dimensional gels, and actin binding proteins can influence the specific structure the filaments will form.
Although a large body of work suggests that F-actin can exist in multiple states, in general, an actin filament has a total rise of 27.3 Å between subunits on adjacent strands and a rotation of 166.15° around the axis. An actin filament is flexible, has a helical repeat every 37 nm, ranges from 5-9 nm in diameter, and has 13 actin subunits between each cross-over point (produced by the ‘crossing over’ of the two long-pitch actin helices). An exception is the case of sperm, where a crosslinker protein, scruin, maintains the actin in a coiled state and extends upon activation. In the acrosomal actin rotation angle of one of the strands is 167.15° while the other strand is tilted more than usual to fit into an asymmetric helix.
The formins are a large family of proteins that facilitate the nucleation of new, unbranched filaments by promoting the interaction between two actin monomers. Under normal circumstances formins are auto-inhibited through structural interactions between the two ends of the protein. However, conformational rearrangements resulting in their activation can be induced through interactions with GTP-bound (active) Rho GTPases. This process remains poorly understood.
The tumor suppressor adenomatous polyposis coli (APC) was shown to bind the formin mDia1 and overcome capping protein- and profilin-mediated suppression of spontaneous actin nucleation, resulting in the initiation of actin filament nucleation and elongation. In the mechanism described, APC is primarily responsible for actin monomer recruitment, whilst mDia1 catalyzes filament elongation. Actin recruitment by APC did not involve capturing F-actin intermediates that had spontaneously formed nor did APC contribute to filament elongation. In this model, once actin polymerization commences the APC-mDia1 complex separates – mDia1 is propelled away from APC along with the growing barbed end of the filament and APC remains attached to the filament at the site of nucleation.
Although a consensus has yet to be reached for the mechanism of formin-mediated nucleation, it is now well-established that activated formins function as dimers and form a donut-shaped complex around terminal actin subunits, orientating themselves toward the (+) end of the actin filament or nucleus. This binding is facilitated by FH2 (formin homology 2) domains within the formin monomers. Next, each formin monomer binds and captures profilin units, which are themselves already bound to G-actin monomers. This interaction is mediated by multiple stretches of polyproline residues within the FH1 domain of formins. This domain is known to range from 15-229 residues, consist of between 35% and 100% proline residues, and contain up to 16 profilin binding sites. Profilin maintains a steady pool of actin monomers by promoting ADP to ATP nucleotide exchange on G-actin. These monomers of ATP-G-actin are then added the growing actin filament. The coupling of formin with the growing end prevents capping and allows continued growth of the filaments.
The first step in actin polymerization is known as ‘nucleation’. This step sees the formation of an actin nucleus, which is essentially a complex of three actin monomers, from which an actin filament may elongate. Although non-muscle cells have a high concentration of G-actin-ATP (~100 μM), pure G-actin monomers fail to nucleate new actin filaments efficiently due to the instability of actin oligomers. Additional factors are therefore required and although the exact mechanisms behind filament nucleation remain to be clearly defined, two models, each involving distinct mechanisms and proteins have been proposed. Importantly, these mechanisms are not mutually exclusive and it maybe be the case that nucleation of actin filaments results from a combination of both mechanisms.
In the first model, known as the ‘tip nucleation model’, members of the formin family of proteins cluster at the plasma membrane and initiate the nucleation of actin filaments. Formin subsequently mediates filament extension with the structural integrity of the filament bundles maintained by fascin cross-linking. The alternative model is known as the “convergent elongation model”. In this model, the Arp2/3 complex, which is more commonly associated with lamellipodia formation but has been found to be critical for filopodia initiation, plays a role. Here, Arp2/3 complex nucleated branches continually develop from the actin filament network located at the leading edge of the lamellipodia. These filaments are proposed to gradually converge, forming a bundle that is secured by facsin cross-linking.