Microfilament



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.

Organization
Actin filaments are assembled in two general types of structures: bundles and networks. Actin-binding proteins dictate the formation of either structure since they cross-link actin filaments in the double-stranded helix.

Bundles
In non-muscle actin bundles, the filaments are held together such that they are parallel to each other by actin-bundling and/or cationic species. Bundles play a role in many cellular processes such as cell division (cytokinesis) and cell movement.

In vitro self-assembly
The thinnest fibers of the cytoskeleton (measuring approximately 7 nm in diameter), microfilaments are formed by the head-to-tail polymerization of actin monomers (also known as globular or G-actin). Actin subunits as part of a fiber at referred to as filamentous actin (or F-actin). Each microfilament is made up of two helical interlaced strands of  subunits. Much like microtubules, actin filaments are polarized, with their fast-growing (+)-ends (also known as barbed ends, because of their appearance in electron micrographs after binding of myosin S1 sub-fragments) and a slow-growing (-)-end (or pointed end, again based on the pattern created by S1 binding).

Filaments elongate approximately 10 times faster at their plus (+) ends than their minus (-) ends. At steady-state, the polymerization rate at the plus end matches the depolymerization rate at the minus end, and microfilaments are said to be treadmilling. A treadmilling filament need not move; even so, there is a net monomer uptake at the (+)-end and a net monomer loss from the (-)-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 plus (+) 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 minus 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 (+)-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.

Microfilament-based motility by actoclampin molecular motors
Intracellular actin cytoskeletal assembly and disassembly are tightly regulated by cell signaling mechanisms. Many signal transduction systems use the actin cytoskeleton as a scaffold holding them at or near the inner face of the peripheral membrane. This subcellular location allows immediate and exquisite responsiveness to transmembrane receptor action and signal-processing enzyme cascades. Because actin monomers must be recycled to sustain high rates of actin-based moltility during chemotaxis, cell signalling is believed to activate cofilin, an actin-filament depolymerizing protein which binds to ADP-rich actin subunits nearest the filament's (-)-end and promotes filament fragmentation, with concomitant depolymerization to liberate actin monomers. The protein profilin enhances the ability of monomers to assemble by stimulating the exchange of actin-bound ADP for solution-phase ATP to yield Actin-ATP and ADP. In most animal cells, monomeric actin is bound to profilin and thymosin-beta4, both of which preferentially bind with one-to-one stoichiometry to ATP-containing monomers. Although thymosin-beta4 is strictly a monomer-sequestering protein, the behavior of profilin is far more complex. Profilin is transferred to the leading edge by virtue of its PIP2 binding site, and profilin also employs its poly-L-proline binding site to dock onto end-tracking proteins. Once bound, Profilin-Actin-ATP is loaded into the monomer-insertion site of actoclampin motors (see below). Another important component in filament formation is the Arp2/3 complex, which binds to the side of an already existing filament (or "mother filament"), where it nucleates the formation of a new actin filament and creates a fan-like branched filament network.

In non-muscle cells, actin filaments are formed at/near membrane surfaces. Their formation and turnover are regulated by many proteins, including
 * Filament end-tracking protein (e.g., formins, VASP, N-WASP)
 * Filament-nucleator known as the Actin-Related Protein-2/3 (or Arp2/3) complex
 * Filament cross-linkers (e.g., α-actinin and fascin)
 * Actin monomer-binding proteins profilin and thymosin-β4
 * Filament (+)-end cappers such as Capping Protein and CapG, etc.
 * Filament-severing proteins like gelsolin
 * (-)-End depolymerizing proteins such as ADF/cofilin

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 (+)-end of each filament attached to the cell's peripheral membrane by means of clamped-filament elongation motors ("actoclampins") formed from a filament (+)-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 (-)-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 (+)-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:
 * 1) The polymerization cofactor profilin and the ATP·actin combine to form a profilin-ATP-actin complex that then binds to the end-tracking unit
 * 2) The cofactor and monomer are transferred to the (+)-end of an actin already clamped filament
 * 3) The tracking unit and cofactor dissociate from the adjacent protofilament, in a step that can be facilitated by ATP hydrolysis energy to modulate the affinity of the cofactor and/or the tracking unit for the filament; and this mechanoenzymatic cycle is then repeated, starting this time on the other sub-filament growth site.

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.