Actin is a highly abundant (10-100 micromolar on average),~42 kDa structural protein found in all eukaryotic cells (except for nematode sperm). With more than 95% conservation in the primary structure, actin is one of the most highly-conserved proteins . The monomeric, globular form of actin, known as G-actin, forms the basic unit for actin filaments. In many cases actin filaments may bundle together with other actin filaments, or, together with their associated motor proteins (e.g. myosin superfamily) form an elaborate network known as the actin cytoskeleton. This occurs primarily at or near the plasma membrane. Consequently a region of high actin filament density is commonly found at the cell periphery and is known as the cell cortex. Actin filaments in the cell cortex determine the shape, stiffness and movement of the cell surface. The actin cytoskeleton also facilitates the transduction of mechanical signals, and can generate the intracellular forces that are required for many cellular functions including cell motility, muscle contraction, cell division, cytokinesis, vesicle and organelle movement and cell signaling. Actin also contributes to the formation and maintenance of cell junctions.
Higher eukaryotes commonly express several isoforms of actin; encoded by a family of related genes. Actin isoforms are divided into three classes (alpha [α], beta [β] and gamma [γ]) according to their isoelectric point. In general, alpha actins are found in muscle (α-skeletal, α-aortic smooth, α-cardiac, and γ2-enteric smooth), whereas beta and gamma isoforms are prominent in non-muscle cells (β- and γ1-cytoplasmic). Early models that described actin filaments were constructed by fitting the filament x-ray crystal structure to the atomic structure of actin monomers  (reviewed in ). More recent models used a number of different approaches . Collectively however these results suggest that when single actin strands form, two asymmetric actin monomers align to form a twofold axis of symmetry ; their subsequent assembly into a filament that is composed of a pair of strands causes a left-handed helical twist when the adjacent subunits are positioned with respect to each other .
Actin filaments are highly dynamic and their polymerization is usually correlated to their disassembly. Generally, actin filament polymerization occurs over three phases: A nucleation phase, an elongation phase and a steady state phase.
During the nucleation phase the formation of a stable ‘actin nucleus’ occurs. This is usually comprised of three actin monomers in complex. In the elongation phase monomers are rapidly added to the filament at the (+ve) or barbed end and this is often facilitated by additional elongation factors such as formin. For this process to occur, the (+) end of the filament must be exposed, and this means removal of *capping protein.
Formins promote the elongation of pre-existing filaments by removing barbed end capping proteins and forming a sleeve around the actin subunits. Formins are also capable of actin nucleation, a process which is spatiotemporally coupled with actin disassembly .
It is well-established that activated formins facilitate elongation as dimers and form a donut-shaped complex around terminal actin subunits, orientating themselves toward the (+) end of the actin filament . Binding, which is facilitated by FH2 (formin homology 2) domains within the formin monomers removes capping protein from the end of the filament and prevents re-capping to allow continued growth of filaments or cross-linked bundles (, reviewed in ).
Formins nucleate and polymerize actin filaments at focal adhesions at a rate of around 0.3 µm/min . Inhibiting formin protein expression results in a decreased filament elongation rate (0.1 µm/min), coupled with abnormal stress fiber morphology and an accumulation of actin binding proteins (e.g. α-actinin ). Ena/VASP proteins support formin-mediated filament elongation by tethering the filaments near sites of active actin assembly .
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 .
Formins could, in theory, contribute to protrusive forces by remaining attached to the barbed end of actin filaments  (reviewed in ). Consistent with this notion, excessive formin activity promotes cell migration. However, the specific mechanisms involved remain unknown given that formin-induced activity does not impact the overall adherence of cells to their substrate, nor does it change the avidity or affinity of cell adhesion receptors (e.g. integrins) .
Profilin binds simultaneously to formin and actin monomers; this interaction tethers multiple profilin-actin complexes near the growing end of actin filaments, which promotes the processive addition of actin subunits . Profilin uses the energy from ATP hydrolysis generated during actin polymerization to facilitate actin assembly . Profilin binds to cytoplasmic ATP-actin monomers better than cytoplasmic ADP-actin monomers .
Profilin has been suggested to generally increase the elongation rate of formin-associated filaments by:
Importantly however, profilin also promotes disassembly of actin filaments by sequestering monomeric G-actin, thereby blocking its association with the barbed ends and promoting its disassembly from the pointed ends of actin filaments . The combined actions of profilin and ADF/cofilin synergize to enhance turnover of actin filaments .
In the steady state phase, the filament dynamics enter a state of equilibrium where monomer disassembly from the (-) end and polymerization at the (+) end is balanced and maintained by a critical concentration of monomers in the cytosol. This steady state assembly and disassembly is known as ‘treadmilling’.
A steady pool of actin monomers must be maintained to enable a polymerization to continue beyond the rapid elongation phase. This is, in part, aided by the actin binding protein profilin, which promotes ADP to ATP nucleotide exchange on G-actin. However, the rate of monomer dissociation from the (-) end of the filament is also important. Dissociation of the subunits ultimately results from ATP hydrolysis, which induces a conformational change in the actin subunit that weakens its association with neighboring subunits (as reviewed in ). The concentration of actin monomers in the cytosol will either favor disassembly, or assembly of the actin filament, and these values are known as the critical concentration (Cc). When the concentration of free subunits exceeds the Cc, filament elongation occurs spontaneously . Importantly, the Cc usually varies between the filament (+) end and the (-) end. At the steady state, which is achieved when the rate of filament polymerization is equally balanced by filament disassembly, the free subunit concentration is higher than the Cc at the (+) end and lower than the Cc at the (-) end. This results in subunits being added to the (+) end and dissociating from the (-).
When the association rate of free ATP-G-actin is greater than the rate of subunit loss, the filament appears to grow, creating a ‘cap’ rich in ATP-subunits . Conversely, when the association rate of free ATP-actin is lower than the rate of subunit loss, the filament is seen to shrink. When the association rate of free ATP-actin is equal to the rate of dissociation at the (-) end, no net growth occurs and this is known as ‘treadmilling’.
Growth and disassembly of actin filaments are tightly controlled by additional proteins. These proteins may either promote actin filament nucleation by stabilizing the actin nucleus, catalyze filament elongation or promote actin treadmilling. Some well established proteins that play such roles include the Arp2/3 complex, which facilitates nucleation of filament branches; profilin, which catalyzes ADP to ATP exchange and ADF/cofilin, which mediates filament disassembly. The cooperation between each component is extensive and each element has an optimal concentration.
ATP-binding on actin subunits modulates the dynamics of filament assembly, with ATP-binding generally favoring intersubunit interactions and thereby filament assembly . The rate of actin addition to filaments depends on the concentration of free ATP-G-actin whereas the rate of subunit loss does not. At high free ATP-G-actin concentrations the rate of addition exceeds the rate of dissociation and this results in actin filament growth.
The addition of ATP-G-actin to the two ends of preexisting actin filaments occurs at very different rates (see Table 1) . The addition of free ATP-G-actin at the (-) end is much lower relative to the (+) end.
The (-) and (+) ends have a different criticial concentration (Cc) for actin filament growth. The Cc is defined as the concentration level of free ATP-G-actin where the rate of addition is balanced by the rate of loss and no net growth occurs at that end. At concentrations above the Cc, actin filament growth occurs, wheras below it, there is a loss of subunits and shrinkage occurs. Any protein that alters the Cc, e.g. profilin, will alter actin filament dynamics.
Toxins such as phalloidins, cytochalasins, latrunculin A, and jasplakinolide are naturally occurring small molecules that bind to actin and alter its polymerization. Phalloidins inhibit actin filament disassembly by locking adjacent actin subunits together, while cytochalasins bind to the barbed end of actin filaments to prevent actin filament assembly and disassembly at that end. Latrunculin A binds to actin monomers to inhibit their polymerization and thus promotes filament disassembly; latrunculin A may also inhibit nucleotide exchange on actin subunits . Jasplakinolides stabilize actin monomers, thereby enhancing filament nucleation and assembly.
Certain actin binding proteins initiate actin filament assembly/disassembly directly, while others influence the ATP binding, the rate of G-actin assembly and the Cc of the filament ends. There are over 60 families of actin-binding proteins (reviewed in ) with the main actin monomer-binding proteins in vertebrate cells being thymosin-β4 and profilin. Thymosin-β4 binds strongly to ATP-actin and prevents its assembly into filaments . Because profilin and thymosin-β4 have overlapping binding sites on actin , profilin must compete with thymosin-β4 during filament assembly . Other examples of actin-binding proteins include: the nucleators spire and the Arp2/3 complex, elongation factors such as formin, actin cross-linking proteins such as fascin or filamin A, nucleation promoting factors such as WASp, and capping protein.