Actin filaments (also known asmicrofilaments) areprotein filaments in thecytoplasm ofeukaryoticcells that form part of thecytoskeleton.[1] They are primarily composed ofpolymers ofactin, but are modified by and interact with numerous otherproteins in the cell. Actin filaments are usually about 7nm in diameter and made up of two strands of actin.[1] Microfilament functions includecytokinesis,amoeboid movement,cell motility, changes in cell shape,endocytosis andexocytosis, cell contractility, and mechanical stability. In inducingcell motility, one end of the actin filament elongates while the other end contracts, presumably bymyosin II molecular motors.[2] Additionally, they function as part ofactomyosin-driven contractile molecular motors, wherein the thin filaments serve as tensile platforms for myosin'sATP-dependent pulling action inmuscle contraction andpseudopod advancement. Microfilaments have a tough, flexible framework which helps the cell in movement.[3]
Actin was first discovered in rabbit skeletal muscle in the mid 1940s byF.B. Straub.[4] Almost 20 years later,H.E. Huxley demonstrated that actin is essential formuscle contraction. The mechanism in which actin creates long filaments was first described in the mid 1980s. Later studies showed that actin has an important role in cell shape, motility, and cytokinesis.
Actin filaments are assembled in two general types of structures: bundles and networks. Bundles can be composed of polar filament arrays, in which all barbed ends point to the same end of the bundle, or non-polar arrays, where the barbed ends point towards both ends. A class ofactin-binding proteins, calledcross-linking proteins, dictate the formation of these structures. Cross-linking proteins determine filament orientation and spacing in the bundles and networks. These structures are regulated by many other classes of actin-binding proteins, including motor proteins, branching proteins, severing proteins, polymerization promoters, and capping proteins.[citation needed]
Measuring approximately 7nm in diameter, microfilaments are the thinnest fibers of the cytoskeleton.[1] They arepolymers ofactin subunits (globular actin, or G-actin), which as part of the fiber are referred to as filamentous actin, or F-actin. Each microfilament is made up of twohelical, interlaced strands of subunits. Much likemicrotubules, actin filaments are polarized.Electron micrographs have provided evidence of their fast-growing barbed-ends and their slow-growing pointed-end. This polarity has been determined by the pattern created by thebinding of myosin S1 fragments: they themselves are subunits of the larger myosin IIprotein complex. The pointed end is commonly referred to as the minus (−) end and the barbed end is referred to as the plus (+) end.[citation needed]
In vitro actin polymerization, starts with the self-association of three G-actin monomers to form atrimer.ATP-bound actin then itself binds the barbed end, and the ATP is subsequentlyhydrolyzed. ATP hydrolysis occurs with ahalf time of about 2 seconds,[5] while the half time for the dissociation of theinorganic phosphate is about 6 minutes.[5] Thisautocatalyzed event reduces the binding strength between neighboring subunits, and thus generally destabilizes the filament.In vivo actin polymerization is catalyzed by a class of filament end-tracking molecular motors known asactoclampins.[6] Evidence suggests that the rate of ATP hydrolysis and the rate of monomer incorporation are strongly coupled.[citation needed]
Subsequently,ADP-actin dissociates slowly from the pointed end, a process significantly accelerated by the actin-binding protein,cofilin. ADP bound cofilin severs ADP-rich regions nearest the (−)-ends. Upon release, the free actin monomer slowly dissociates from ADP, which in turn rapidly binds to the free ATPdiffusing in thecytosol, 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 asCapZ prevent the addition or loss of monomers at the filament end where actin turnover is unfavorable, such as in the muscle apparatus.[citation needed]
Actin polymerization together with capping proteins were recently used to control the 3-dimensional growth of protein filament so as to perform 3D topologies useful in technology and the making of electrical interconnect. Electrical conductivity is obtained by metallisation of the protein 3D structure.[7][8]
As a result of ATP hydrolysis, filaments elongate approximately 10 times faster at their barbed ends than their pointed ends. Atsteady-state, the polymerization rate at the barbed end matches the depolymerization rate at the pointed end, and microfilaments are said to betreadmilling. Treadmilling results in elongation in the barbed end and shortening in the pointed-end, so that the filament in total moves. Since both processes are energetically favorable, this means force is generated, the energy ultimately coming from ATP.[2]
Intracellular actin cytoskeletal assembly and disassembly are tightly regulated by cell signaling mechanisms. Manysignal transduction systems use the actin cytoskeleton as a scaffold, holding them at or near the inner face of the peripheralmembrane. This subcellular location allows immediate responsiveness to transmembrane receptor action and the resulting cascade of signal-processing enzymes.[citation needed]
Because actin monomers must be recycled to sustain high rates of actin-based motility duringchemotaxis, cell signalling is believed to activate cofilin, the actin-filament depolymerizing protein which binds to ADP-rich actin subunits nearest the filament's pointed-end and promotes filament fragmentation, with concomitant depolymerization in order to liberate actin monomers. In most animal cells, monomeric actin is bound toprofilin andthymosin beta-4, both of which preferentially bind with one-to-one stoichiometry to ATP-containing monomers. Although thymosin beta-4 is strictly a monomer-sequestering protein, the behavior of profilin is far more complex. 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. Profilin is transferred to the leading edge by virtue of itsPIP2 binding site, and it 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.[citation needed]
Another important component in filament formation is theArp2/3 complex, which binds to the side of an already existing filament (or "mother filament"), where it nucleates the formation of a new daughter filament at a 70-degree angle relative to the mother filament, effecting a fan-like branched filament network.[9]
Specialized unique actin cytoskeletal structures are found adjacent to the plasma membrane. Four remarkable examples includered blood cells,human embryonic kidney cells,neurons, andsperm cells. In red blood cells, aspectrin-actinhexagonal lattice is formed by interconnected short actin filaments.[10] In human embryonic kidney cells, the cortical actin forms a scale-freefractal structure.[11] First found in neuronalaxons, actin forms periodic rings that are stabilized by spectrin and adducin[12][13] – and this ring structure was then found by He et al 2016 to occur in almost every neuronal type andglial cells, across seemingly every animal taxon includingCaenorhabditis elegans,Drosophila,Gallus gallus andMus musculus.[14] And in mammalian sperm, actin forms ahelical structure in the midpiece, i.e., the first segment of theflagellum.[15]
In non-muscle cells, actin filaments are formed close to membrane surfaces. Their formation and turnover are regulated by many proteins, including:[citation needed]
The actin filament network in non-muscle cells is highly dynamic. 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, the above-mentioned "actoclampins", formed from a filament barbed-end and a clamping protein (formins, VASP, Mena, WASP, and N-WASP).[16] The primary substrate for these elongation motors is profilin-actin-ATP complex which is directly transferred to elongating filament ends.[17] 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 filopodia a parallel array of filaments is formed.[citation needed]
Myosinmotors are intracellular ATP-dependent enzymes that bind to and move along actin filaments. Various classes of myosin motors have very different behaviors, including exerting tension in the cell and transporting cargo vesicles.[citation needed]
One proposed model suggests the existence of actin filament barbed-end-trackingmolecular motors termed "actoclampins".[18] The proposed actoclampins generate the propulsive forces needed for actin-based motility oflamellipodia,filopodia, invadipodia,dendritic spines,intracellularvesicles, andmotile processes inendocytosis,exocytosis, podosome formation, andphagocytosis. Actoclampins also propel such intracellularpathogens asListeria monocytogenes,Shigella flexneri,Vaccinia andRickettsia. When assembled under suitable conditions, these end-tracking molecular motors can also propelbiomimetic particles.[citation needed]
The termactoclampin is derived fromacto- to indicate the involvement of an actin filament, as in actomyosin, andclamp to indicate a clasping device used for strengthening flexible or 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 aclampin.[citation needed]
Dickinson and Purich recognized that promptATP hydrolysis could explain the forces achieved during actin-based motility.[16] They proposed a simplemechano-enzymatic sequence known as theLock, 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, whereuponATP 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.[citation needed]
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.[16][18][19] 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.[citation needed]
Some actoclampins (e.g., those involving Ena/VASP proteins, WASP, and N-WASP) apparently require Arp2/3-mediated filament initiation to form theactin 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. The Arp2/3 complex binds to the side of the mother filament, forming a Y-shaped branch having a 70-degree angle with respect to thelongitudinal 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 gate. Whether ATP hydrolysis may be required for nucleation and/or Y-branch release is a matter under active investigation.[citation needed]
