Actin monomer, depicted above in two display modes, has subdomains 1-4. A simplified cartoon is at right. ATP binds, along with Mg ++, within a deep cleft between subdomains 2 & 4.
Actin can hydrolyze its bound ATP ADP + P i, releasing P i. The actin monomer can exchange bound ADP for ATP. The conformation of actin is different, depending on whether ATP or ADP is in the nucleotide-binding site.
G-actin (globular actin), with bound ATP, can polymerize to form F-actin (filamentous). F-actin may hydrolyze bound ATP ADP + P i & release P i. ADP release from the filament does not occur because the cleft opening is blocked. ADP/ATP exchange: G-actin can release ADP & bind ATP, which is usually present at higher concentration than ADP in the cytosol.
Actin filaments have polarity. The actin monomers all orient with their cleft toward the same end of the filament, called the minus end. The diagram above is oversimplified. Actin monomers spiral around the axis of the filament, with a structure resembling a double helix. See diagram in Biomachina websitewebsite
The polarity of actin filaments may be visualized by decoration with globular heads (S1) cleaved off of myosin by proteases. Bound myosin heads cause an appearance of arrowheads in electron micrographs. See images in website of the Heuser Lab.website
In one experiment, short actin filaments were decorated with myosin heads. After removal of excess unbound myosin, the concentration of G-actin was increased, to promote further actin polymerization. Filament growth at one end, designated plus (+), exceeded growth at the other end, designated minus ( ). In electron micrographs, bound myosin heads appear as arrowheads pointing toward the negative end of the filament. Barbed ends orient toward the plus end.
Actin filaments may undergo treadmilling, in which filament length remains approximately constant, while actin monomers add at the (+) end and dissociate from the ( ) end. This has been monitored using brief exposure to labeled actin monomers (pulse labeling).
Capping proteins bind at the ends of actin filaments. Different capping proteins may either stabilize an actin filament or promote disassembly. They may have a role in determining filament length. Examples: Tropomodulins cap the minus end, preventing dissociation of actin monomers. CapZ capping protein binds to the plus end, inhibiting polymerization. If actin monomers continue to dissociate from the minus end, the actin filament will shrink.
Two toxins that have been useful experimentally: Cytochalasins (from fungi) bind to the (+) end of F-actin and block subunit addition. Depolymerization at the ( ) end may cause loss of the filament. Phalloidin (from Amanita mushroom) binds along the sides of actin filaments, stabilizing them. Phalloidin labeled with a fluorescent chromophore is often used to visualize actin filaments by fluorescence microscopy.
Some actin-binding proteins such as -actinin, villin & fimbrin bind actin filaments into parallel bundles. Depending on the length of a cross-linking protein, or the distance between actin-binding domains, actin filaments in parallel bundles may be held close, or may be far enough apart to allow interaction with other proteins, e.g., myosin. Cross-linking proteins organize actin filaments into bundles or networks. Actin-binding domains of several cross-linking proteins (e.g., filamin, -actinin, spectrin, dystrophin & fimbrin) are homologous. Most cross-linking proteins are dimeric or have 2 actin-binding domains.
Filamins organize actin filaments into loose networks that give some areas of the cytosol a gel-like consistency. Filamins may also have scaffolding roles relating to their ability to bind constituents of signal pathways such as plasma membrane receptors, calmodulin, caveolin, protein kinase C, transcription factors, etc. Filamins dimerize, through antiparallel association of their C-terminal domains, to form V-shaped cross-linking proteins that have a flexible shape due to hinge regions.
Spectrin is an actin-binding protein that forms an elongated tetrameric complex having an actin-binding domain at each end. With short actin filaments, spectrin forms a cytoskeletal network on the cytosolic surface of the plasma membrane of erythrocytes and some other cells. For a diagram see a website of L. Backman.website
Cell structures that involve actin: Filopodia (microspikes) are long, thin and transient processes that extend out from the cell surface. Bundles of parallel actin filaments, with plus ends oriented toward the filopodial tip, are cross-linked by a small actin-binding protein such as fascin. The closely spaced actin filaments provide stiffness. Microvilli are shorter & more numerous protrusions of the cell surface found in some cells. Tightly bundled actin filaments within these structures also have their plus ends oriented toward the tip. Small cross-linking proteins such as fimbrin and villin bind actin filaments together within microvilli.
Lamellipodia are thin but broad projections at the edge of a mobile cell. Lamellipodia are dynamic structures, constantly changing shape. See Movie on lamellipodial action in wound closure.Movie Lamellipodia, at least in some motile cells, have been shown to contain extensively branched arrays of actin filaments, oriented with their plus (barbed) ends toward the plasma membrane. Forward extension of a lamellipodium occurs by growth of actin filaments adjacent to the plasma membrane.
Stress fibers form when a cell makes stable connections to a substrate. Bundles of actin filaments extend from the cell surface through the cytosol. The actin filaments, whose plus ends are oriented toward the cell surface on opposite sides of the cell, may overlap in more interior regions of a cell in anti-parallel arrays. Myosin mediates sliding of anti-parallel actin filaments during contraction of stress fibers. -Actinin may cross-link actin filaments within stress fibers.
Some cells have a cytoskeletal network just inside the plasma membrane that includes actin along with various other proteins such as spectrin. This cytoskeleton has a role in maintaining cell shape. An example is found in erythrocytes. Actin filaments have an essential role in the contractile ring responsible for cytokinesis at the end of mitosis in animal cells. Actin is found in the cell nucleus as well as in the cytoplasm. Recent data indicate involvement of actin in regulation of gene transcription.
Arp2/3 complex includes 2 actin-related proteins, Arp2 & Arp3, plus 5 smaller proteins. When activated by a nucleation promoting factor (NPF), Arp2/3 complex binds to the side of an existing actin filament and nucleates assembly of a new filament branch. The resulting branch structure is Y-shaped. In this oversimplified diagram, Arp2 & Arp3 are shown forming the start of a new branch of double-helical F-actin. Nucleation of actin polymerization: Arp2/3 nucleates actin polymerization in lamellipodia.
It has been argued that the network of short, branching actin filaments seen in lamellipodia of some cell types could be more effective in pushing the leading edge forward than unbranched filaments, given the flexibility of actin filaments. WebsiteWebsite with movies & micrographs. At the leading edge of a lamellipodium, plus end capping proteins may keep actin filaments short, while Arp2/3 keeps initiating new branches to propel the edge of the cell forward.
Further back from the leading edge, actin-destabilizing proteins, e.g., cofilin & gelsolin (to be discussed), would promote loss of actin monomers from the minus end. The continuous plus-end filament growth at the leading edge, and minus-end disassembly behind, show up as treadmilling of labeled actin monomers. See a movie (select Fig 10).movie
Formins nucleate formation of unbranched actin filaments, such as those in stress fibers. Formins are found at the plus ends of actin filaments. Formin is said to be processive, because it remains bound to the plus end of an actin filament as actin monomers are added at the plus end. The continued presence of formin prevents binding of plus-end capping proteins that would inhibit filament growth.
Each formin includes an actin-binding FH2 domain that dimerizes to form a ring-like structure with flexible links. Models have been proposed involving "stair stepping" by the dimeric formin to explain its ability to remain at the plus end as actin monomers are added. E.g., it has been proposed that one FH2 domain of the dimeric formin may shift to an "open" conformation allowing entry of an actin monomer as the other FH2 domain binds to the most recently added actin subunit. See a movie (Choose supplemental materials).movie
Other formin domains: Another actin-binding domain (FH1) binds monomeric actin complexed with profilin (to be discusssed). This may increase the effective concentration of monomeric actin adjacent to the polymerization site. Regulatory domains of formins allow for autoinhibition that is turned off during activation of actin polymerization by the GTP-binding signal protein Rho (to be discussed). See a diagram (Fig. 1).diagram
Integrins mediate adhesion of cells to the extracellular matrix as well as to other cells. Cytosolic domains of integrins bind to adaptor proteins (e.g., -actinin, talin, filamin) that link integrins to elements of the cytoskeleton such as actin filaments. Integrins: heterodimeric cell surface receptors. Each of the 2 integrin subunits, designated & , is a single-pass transmembrane protein.
Different combinations of & subunits yield a variety of integrins with different binding specificity. E.g.: Extracellular domain of 1 1 integrin binds collagen. Extracellular domain of 5 1 integrin binds fibronectin. Extracellular ligands bind at the / subunit interface. Extracellular domains of both & integrin subunits contribute residues to the ligand binding site. There are multiple isoforms of & subunits.
Integrins mediate dynamic connections between the actin cytoskeleton inside a cell and constituents of the extracellular matrix. Moving cells make & break contacts with the matrix, whereas stationary cells may form more stable complexes with extracellular constituents.
Integrins have signaling as well as adhesive roles. Outside-in signaling: Binding of ligands by extracellular domains may generate conformational changes that affect interaction of integrins with intracellular cytoskeletal and signal proteins. Inside-out signaling: The affinity of integrins for extracellular ligands is subject to regulation by cell signals. The inactive integrin has a bent over conformation, while in the fully activated state globular ligand binding domains extend out maximally from the cell surface. See diagrams in a website of the Walz lab at Harvard.website
In focal adhesions stress fibers attach via adapter proteins to plasma membrane integrins. The adapter proteins that link actin filaments to cytosolic domains of integrins include -actinin & talin. With extracellular domains of the integrins linked to matrix proteins, a cell is firmly attached to the external matrix.
Gelsolin functions in gel sol transitions in the cytosol. When activated by Ca ++, gelsolin, severs an actin filament and caps the (+) end, blocking filament regrowth. Actin filaments become kinked prior to being severed by gelsolin.
Gelsolin may also function to promote forward extension of a lamellipodium. By severing actin filaments, gelsolin contributes to the development of the branched actin filament networks that grow to propel forward the plasma membrane at the leading edge. Gelsolin in the absence of Ca ++ does not bind actin. Ca ++ causes a large conformational change in gelsolin that exposes an actin-binding site. Actin contributes a Glu carboxyl group to one of the two Ca ++ -binding sites.
binds along the side of the actin monomer and in a cleft between actin subdomains 1 & 3, at the plus end. The hydrophobic cleft between actin subdomains 1 & 3 is a common site of interaction with actin-binding proteins. Subdomain 2 of actin itself binds to this cleft of the adjacent monomer in F-actin. G-actin shown complexed with the C-terminal half of gelsolin, with bound Ca ++. Gelsolin, which interacts also with filamentous actin,
Cofilin is a member of the ADF (actin depolymerizing factor) protein family. Cofilin binds to actin-ADP along the sides of actin filaments, distorting the helical twist. Under some conditions cofilin can sever actin filaments. Cofilin also promotes dissociation of G-actin-ADP (as a complex with cofilin) from minus ends of actin filaments.
Cofilin may then bind to G-actin-ADP and inhibit ADP/ATP exchange. This would inhibit actin re-polymerization. Phosphorylation of cofilin causes it to dissociate from G-actin, which can then undergo ADP/ATP exchange and add to the (+) end of F-actin. Actin polymerization in some cases may be triggered by signal cascades leading to phosphorylation of cofilin.
Twinfilin is a protein structurally related to cofilin that binds G-actin-ADP, and may have a role in sequestering actin monomers. Thymosin 4 is a small protein (5 kDa) that also forms a 1:1 complex with G-actin. Thymosin is proposed to “buffer” the concentration of free actin, by maintaining a pool of monomeric actin. An increase in the concentration of thymosin 4 may promote depolymerization of F-actin, by lowering the concentration of free G-actin.
Profilin has a role in regulating actin polymerization. Profilin forms a 1:1 complex with G-actin. Profilin binding at the plus end, opposite the nucleotide-binding cleft, alters the conformation of G-actin, making its nucleotide-binding site more open to the cytosol. This promotes ATP/ADP exchange.
The stimulation by profilin of ATP/ADP exchange increases the local concentration of G-actin-ATP, the form able to polymerize. Profilin may sequester actin monomers. Localized release of G-actin-ATP by profilin may promote actin polymerization. Usually profilin promotes actin polymerization. It may function as a carrier, donating the actin monomer to the plus end of a filament. Because profilin binds at the plus end of an actin monomer, the actin monomer's minus end is available for addition to the plus end of an existing actin filament. ++
Derivatives of the membrane lipid phosphatidylinositol are involved in signal cascades. Signal-activated kinases convert phosphatidylinositol to PIP 2 (phosphatidylinositol-4,5-bisphosphate). Regulation of assembly and disassembly of the actin cytoskeleton is very complex.
Phospholipase C, activated by a signal cascade, catalyzes cleavage of PIP 2 to yield the signal molecules diacylglycerol & inositol trisphosphate (IP 3 )
E.g., regulated formation & cleavage of PIP 2 can affect the concentration of profilin, and hence actin polymerization, adjacent to the plasma membrane. In addition to its role in generating the second messengers diacylglycerol and IP 3, PIP 2 formation and hydrolysis can affect the actin cytoskeleton.
Signal-activated PIP 2 hydrolysis releases profilin, which may bind G-actin and promote ADP/ATP exchange. The increase in G-actin-ATP promotes actin polymerization adjacent to the plasma membrane. PIP 2 binds profilin at the cytosolic surface of the plasma membrane. This prevents profilin-actin interaction.
Some actin severing and capping proteins also bind to PIP 2, including gelsolin and cofilin. Filament severing can be a mechanism for increasing the number of plus ends to which actin can polymerize. Binding to PIP 2 inhibits gelsolin and cofilin, and sequesters them near the cell surface. Their regulated release can affect formation of lamellipodia or forward movement of a cell.
WASP/Scar proteins have domains that bind & activate Arp2/3, plus domains that recognize & bind to signaling factors that may be locally generated in a cell. Thus WASP/Scar proteins may determine where in a cell actin polymerization will occur. Some WASP proteins are activated by binding to proteins of the Rho family (see below) and/or to PIP 2. Nucleation promoting factors that activate the Arp2/3 complex include proteins called WASP & Scar (WAVE). The genetic disease Wiskott- Aldrich Syndrome gave WASP its name.
Some pathogens utilize a host cell's actin to move around in an infected cell, & for transmission to other cells. They move by growth of actin tails. In Listeria, a bacterial cell-surface protein homologous to WASP activates the Arp2/3 complex. Shigella has a cell-surface protein that binds WASP. Vaccinia virus has a surface protein that, when phosphorylated, binds via adaptor proteins to WASP. Salmonella encodes actin-binding proteins that appear to directly nucleate actin filament growth. Website Website with movies showing actin-based movement of Listeria and Shigella within cells, maintained by J. Theriot at Stanford.
Rho is a family of small GTP-binding proteins that regulate the actin cytoskeleton. Some members of the Rho family: Rac activates formation of lamellipodia, in part through activation of WASP. Cdc42 activates formation of filopodia, in part through activation of the WASP family protein Scar (WAVE). Rho activates formation of focal adhesions & stress fibers, in part through activation of formins. In each case the active form of the Rho family protein has bound GTP.
Rho protein activation: GEFs (Guanine nucleotide exchange proteins) of the Dbl family promote GDP release with binding of GTP. Dbl GEFs are activated by signal cascades initiated via plasma membrane receptors, e.g., cytokine, growth factor or cell adhesion receptors. Rho protein downregulation: GAPs (GTPase activating proteins) facilitate GTP hydrolysis by Rho. GDIs (Guanine nucleotide dissociation inhibitors) bind to Rho & prevent GDP/GTP exchange.
In addition to activating formins to promote actin filament growth, Rho-GTP promotes myosin-actin interactions essential for development & contraction of stress fibers, through its activation of ROCK (Rho Kinase). ROCK phosphorylates (& inhibits dephosphorylation of) myosin II light chains. Light chain phosphorylation promotes interaction of myosin with actin filaments.
Other Kinases that regulate formation or disassembly of focal adhesions include: Focal Adhesion Kinase (FAK) has a role in modulating assembly of focal adhesions in response to tension exerted by the cytoskeleton on attachments to the extracellular substrate via integrins. FAK acts through various downstream effectors, including constituents of MAP Kinase signal pathways. Integrin-Linked Kinase (ILK) binds to plasma membrane integrins and to various actin-binding proteins, thereby mediating attachment of actin to the plasma membrane in focal adhesions. ILK also phosphorylates & activates Protein Kinase B, an enzyme with many regulatory roles.
Proteins of the ERM family (ezrin, radixin & moesin) provide regulated linkage of actin filaments to the plasma membrane in some cells. ERM proteins bind to actin and bind to cytosolic domains of plasma membrane integral proteins. They are regulated by signal-activated phosphorylation and by interaction with PIP 2.
Calpains are Ca ++ -activated cysteine proteases that regulate cell adhesion. These intracellular proteases cleave constituents of stress fibers & focal adhesions during activation of cell motility. Proteins cleaved by calpains include: actin-binding proteins such as -actinin, filamin, talin and spectrin subunits of plasma membrane integrins the ERM protein ezrin. In addition to being activated by Ca ++, calpains are regulated by phosphorylation and are subject to inhibition by a protein calpastatin.