Microfilaments (or actin filaments) are the thinnest filaments of the cytoskeleton found in the cytoplasm of all eukaryotic cells. These linear polymers of actin subunits are flexible and relatively strong, resisting buckling by multi-piconewton compressive forces and filament fracture by nanonewton tensile forces. Microfilaments are highly versatile, functioning in (a) actoclampin-driven expansile molecular motors, where each elongating filament harnesses the hydrolysis energy of its "on-board" ATP to drive actoclampin end-tracking motors to propel cell crawling, ameboid movement, and changes in cell shape, and (b) actomyosin-driven contractile molecular motors, where the thin filaments serve as tensile platforms for myosin's ATP hydrolysis-dependent pulling action in muscle contraction and uropod advancement.
Filaments elongate approximately 10 times faster at their barbed ends than their pointed ends. At steady-state, the polymerization rate at the barbed end matches the depolymerization rate at the pointed end, and microfilaments are said to be treadmilling. A treadmilling filament need not move; even so, there is a net monomer uptake at the barbed-end and a net monomer loss from the pointed-end, such that the overall length a treadmilling microfilament does not change. Notably, no mechanical force is generated by treadmilling.
In vitro actin polymerization, nucleation, starts with the self-association of three G-actin monomers to form a trimer. ATP-actin then binds the barbed end, and the ATP is subsequently hydrolyzed with a half time of about 2 seconds and the inorganic phosphate released with a half-time of about 6 minutes, which reduces the binding strength between neighboring units and generally destabilizes the filament. In vivo actin polymerization is catalyzed by a new class of filament end-tracking molecular motors known as actoclampins (see next section). Recent evidence suggests that ATP hydrolysis can be prompt in such cases (i.e., the rate of monomer incorporation is matched by the rate of ATP hydrolysis).
ADP-actin dissociates slowly from the pointed end, but this process is greatly accelerated by ADP-cofilin, which severs ADP-rich regions nearest the (–)-ends. Upon release, ADP-actin undergoes exchange of its bound ADP for solution-phase ATP, thereby forming the ATP-actin monomeric units needed for further barbed-end filament elongation. This rapid turnover is important for the cell's movement. End-capping proteins such as CapZ prevent the addition or loss of monomers at the filament end where actin turnover is unfavourable like in the muscle apparatus.
In non-muscle cells, actin filaments are formed at/near membrane surfaces. Their formation and turnover are regulated by many proteins, including
The actin filament network in non-muscle cells is highly dynamic. As first proposed by Dickinson & Purich (Biophysical Journal 92: 622-631), the actin filament network is arranged with the barbed-end of each filament attached to the cell's peripheral membrane by means of clamped-filament elongation motors ("actoclampins") formed from a filament barbed-end and a clamping protein (formins, VASP, Mena, WASP, and N-WASP). The primary substrate for these elongation motors is Profilin-Actin-ATP complex which is directly transferred to elongating filament ends (Dickinson, Southwick & Purich, 2002). The pointed-end of each filament is oriented toward the cell's interior. In the case of lamellipodial growth, the Arp2/3 complex generates a branched network, and in filopods, a parallel array of filaments is formed.
Actoclampins are the actin filament barbed-end-tracking molecular motors that generate the propulsive forces needed for actin-based motility of lamellipodia, filopodia, invadipodia, dendritic spines, intracellular vesicles, and motile processes in endocytosis, exocytosis, podosome formation, and phagocytosis. Actoclampin motors also propel such intracellular pathogens as Listeria monocytogenes, Shigella flexneri, Vaccinia and Rickettsia. When assembled under suitable conditions, these end-tracking molecular motors can also propel biomimetic particles.
The term actoclampin is derived from acto- to indicate the involvement of an actin filament, as in actomyosin, and clamp to indicate a clasping device used for strengthening flexible/moving objects and for securely fastening two or more components, followed by the suffix -in to indicate its protein origin. An actin filament end-tracking protein may thus be termed a clampin.
Dickinson and Purich (2002) recognized that prompt ATP hydrolysis could explain the forces achieved during actin-based motility. They proposed a simple mechanoenzymatic sequence known as the Lock, Load & Fire Model, in which an end-tracking protein remains tightly bound ("locked" or clamped) onto the end of one sub-filament of the double-stranded actin filament. After binding to Glycyl-Prolyl-Prolyl-Prolyl-Prolyl-Prolyl-registers on tracker proteins, Profilin-ATP-actin is delivered ("loaded") to the unclamped end of the other sub-filament, whereupon ATP within the already clamped terminal subunit of the other subfragment is hydrolyzed ("fired"), providing the energy needed to release that arm of the end-tracker, which then can bind another Profilin-ATP-actin to begin a new monomer-addition round.
The following steps describe one force-generating cycle of an actoclampin molecular motor:
When operating with the benefit of ATP hydrolysis, AC motors generate per-filament forces of 8–9 pN, which is far greater than the per-filament limit of 1–2 pN for motors operating without ATP hydrolysis (Dickinson and Purich, 2002, 2006; Dickinson, Caro and Purich, 2004). The term actoclampin is generic and applies to all actin filament end-tracking molecular motors, irrespective of whether they are driven actively by an ATP-activated mechanism or passively.
Some actoclampins (e.g., those involving Ena/VASP proteins, WASP, and N-WASP) apparently require Arp2/3-mediated filament initiation to form the actin polymerization nucleus that is then "loaded" onto the end-tracker before processive motility can commence. To generate a new filament, Arp2/3 requires a "mother" filament, monomeric ATP-actin, and an activating domain from Listeria ActA or the VCA region of N-WASP. Ther Arp2/3 complex binds to the side of the mother filament, forming a Y-shaped branch having a 70 degree angle with respect to the longitudinal axis of the mother filament. Then upon activation by ActA or VCA, the Arp complex is believed to undergo a major conformational change, bringing its two actin-related protein subunits near enough to each other to generate a new filament gat. Whether ATP hydrolysis may be required for nucleation and/or Y-branch release is a matter under active investigation.