How do cells recruit actin monomers from this buffered storage pool and use them for polymerization? The answer depends on another monomer-binding protein called profilin. Profilin binds to the face of the actin monomer opposite the ATP-binding cleft, blocking the side of the monomer that would normally associate with the filament minus end, while leaving exposed the site on the monomer that binds to the plus end (Figure 16–15). When the profilin–actin complex binds a free plus end, a conformational change in actin reduces its affinity for profilin and the profilin falls off, leaving the actin filament one subunit longer. Profilin competes with thymosin for binding to individual actin monomers. Thus, by regulating the local activity of profilin, cells can control the movement of actin subunits from the sequestered thymosin-bound pool onto filament plus ends.
Several mechanisms regulate profilin activity, including profilin phosphorylation and profilin binding to inositol phospholipids. These mechanisms can define the sites where profilin acts. For example, profilin is required for filament assembly at the plasma membrane, where it is recruited by an interaction with acidic membrane phospholipids. At this location, extracellular signals can activate profilin to produce local actin polymerization and the extension of actin-rich motile structures such as filopodia and lamellipodia.
In addition to the availability of active actin subunits, a second prerequisite for cellular actin polymerization is filament nucleation. Proteins that contain actin monomer binding motifs linked in tandem mediate the simplest mechanism of filament nucleation. These actin-nucleating proteins bring several actin subunits together to form a seed. In most cases, actin nucleation is catalyzed by one of two different types of factors: the Arp 2/3 complex or the formins. The first of these is a complex of proteins that includes two actin-related proteins, or ARPs, each of which is about 45% identical to actin. The Arp 2/3 complex nucleates actin filament growth from the minus end, allowing rapid elongation at the plus end (Figure 16–16A and B). The complex can attach to the side of another actin filament while remaining bound to the minus end of the filament that it has nucleated, thereby building individual filaments into a treelike web (Figure 16–16C and D).
Formins are dimeric proteins that nucleate the growth of straight, unbranched filaments that can be cross-linked by other proteins to form parallel bundles. Each formin subunit has a binding site for monomeric actin, and the formin dimer appears to nucleate actin filament polymerization by capturing two monomers.
Figure 16–15 Effects of thymosin and profilin on actin polymerization. An actin monomer bound to thymosin is sterically prevented from binding to and elongating the plus end of an actin filament (left). An actin monomer bound to profilin, on the other hand, is capable of elongating a filament (right). Thymosin and profilin cannot both bind to a single actin monomer at the same time. In a cell in which most of the actin monomer is bound to thymosin, the activation of a small amount of profilin can produce rapid filament assembly. As indicated (bottom), profilin binds to actin monomers that are transiently released from the thymosin-bound monomer pool, shuttles them onto the plus ends of actin filaments, and is then released and recycled for further rounds of filament elongation.
As the newly nucleated filament grows, the formin dimer remains associated with the rapidly growing plus end while still allowing the addition of new subunits at that end (Figure 16–17). This mechanism of filament assembly is clearly different from that used by the Arp 2/3 complex, which remains stably bound to the filament minus end, preventing subunit addition or loss at that end. Formin-dependent actin filament growth is strongly enhanced by the association of actin monomers with profilin (Figure 16–18).