Biochemistry of Metabolism - Cell Biology

Actin Cytoskeleton

Contents of this page:
Actin structure & polymerization

Capping & crosslinking proteins
Cell structures that involve actin

Nucleation of actin filament formation
Integrins & focal adhesions
Regulation of assembly and disassembly of actin filaments

Note: For this topic, references will be given to page numbers in the textbook Molecular Biology of the Cell by Alberts et al. (A). Myosin will be discussed separately.

Actin monomer, depicted above left in two display modes, has subdomains designated 1-4. A simplified cartoon is above right. ATP binds, along with Mg++, within a deep cleft between subdomains 2 and 4.

Actin can hydrolyze its bound ATP to ADP + Pi, releasing Pi. The actin monomer can exchange bound ADP for ATP. The conformation of actin is different, depending on whether there is ATP or ADP in the nucleotide-binding site.

G-actin (globular actin) with bound ATP can polymerize, to form F-actin (filamentous actin).

F-actin may hydrolyze its bound ATP to ADP + Pi and release Pi. ADP release from the filament does not occur because the cleft opening is blocked.

ADP/ATP exchange: G-actin can release ADP and bind ATP, which is usually present in the cytosol at higher concentration than ADP.

Actin filaments have polarity. The actin monomers all orient with their cleft toward the same end of the filament (designated the minus end). The diagram at right is very oversimplified. Actin monomers spiral around the axis of the filament, with a structure resembling a double helix. See diagrams in A p. 916 and in the Biomachina website.

The polarity of actin filaments may be visualized by decorating the filaments with globular heads (designated 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.

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 that 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 of arrowheads 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. For example:
  • 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:

Cross-linking proteins organize actin filaments into bundles or networks. Actin-binding domains of several of the cross-linking proteins (e.g., filamin, a-actinin, spectrin, dystrophin and fimbrin) are homologous. Most cross-linking proteins are dimeric or have 2 actin-binding domains. See A p. 940-943.

Some actin-binding proteins such as a-actinin, villin and 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 together, or may be far enough apart to allow interaction with other proteins such as myosin.

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. 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.

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. See also A p. 603, 940.

Cell structures that involve actin (selected examples):

Nucleation of actin polymerization

Arp2/3 nucleates actin polymerization in lamellipodia. The Arp2/3 complex includes two actin-related proteins, Arp2 and Arp3, plus five 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 the oversimplified diagram at right, Arp2 & Arp3 are shown forming the start of a new branch of double-helical F-actin. See also A p. 933 & 974.

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. 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.

A website of the Borisy lab includes additional movies and electron micrographs depicting the dendritic (branched) organization of actin filaments in lamellipodia of keratinocytes, and movements of these cells. See especially movie sequence #2.

Further back from the leading edge, actin-destabilizing proteins such as cofilin and  gelsolin (see below) 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, as shown in a movie (select Fig 10).

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., 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 linked to a recent review article. Choose supplemental materials. (Requires subscription to Annual Review of Biochemistry.)

Other domains of formin include another actin-binding domain (FH1) that binds monomeric actin complexed with profilin (see below). 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 by the GTP-binding signal protein Rho, discussed below.
For a diagram of formin domain structure see the following web-based review (Fig. 1). (Requires subscription to Annual Review of Biochemistry.)

Integrins are heterodimeric cell surface receptors. Each of the two integrin subunits, designated a and b, is a single-pass transmembrane protein. Diagram at right and see A p. 1113. 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., a-actinin, talin, filamin) that link integrins to elements of the cytoskeleton such as actin filaments.

Extracellular ligands bind at the a/b subunit interface. Extracellular domains of both a & b integrin subunits contribute residues to the ligand binding site.

There are multiple isoforms of a & b subunits. Different combinations of a & b subunits yield a variety of integrins with different binding specificity. For example, the extracellular domain of a1b1 integrin binds collagen, while the extracellular domain of a5b1 integrin binds fibronectin.

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.

The inactive integrin has a bent over conformation, while in the fully activated state the globular ligand binding domains extend out maximally from the cell surface. See diagrams in a website of the Walz lab at Harvard.

In focal adhesions, stress fibers attach via adapter proteins to plasma membrane integrins. The adapter proteins that link stress fibers to integrins include a-actinin and talin. With extracellular domains of the integrins linked to matrix proteins, a cell is firmly attached to the external matrix. 

Regulation of assembly and disassembly of the actin cytoskeleton is very complex. Some examples of regulatory mechanisms are given below.

Gelsolin functions in gel sol transitions within 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.

G-actin is shown complexed with the C-terminal half of gelsolin, with bound Ca++.

Gelsolin, which interacts also with filamentous actin, binds along the side of the actin monomer and in a cleft between actin subdomains 1 and 3, at the plus end. The hydrophobic cleft between actin subdomains 1 and 3 (see figure above) is a common site of interaction with actin-binding proteins. Subdomain 2 of actin itself binds to this same cleft of the adjacent monomer in F-actin.

Chime Exercise: Explore at right the actin-gelsolin-Ca++ complex.


Actin-Gelsolin

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 of F-actin. Under some conditions cofilin can sever actin filaments.

Cofilin also promotes dissociation of G-actin-ADP (as a complex with cofilin) from the minus end 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 also binds G-actin-ADP, and may have a role in sequestering actin monomers.

Thymosin b4, a small protein (5 kDa) 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 b4, 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.

View below, by Chime, the bovine actin-profilin complex. This PDB file 2BTF, is of data by C. E. Schutt, J. C. Myslik, M. D. Rozycki, & N. C. W. Goonesekere, 1994.

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Actin-Profilin - Recommended display options:

Display as cartoon and color chain to distinguish actin (blue) & profilin (green).

Select hetero - ligand, display as spacefill, & color CPK, to visualize ATP, bound to actin with the cation strontium (in place of Mg++). The single atom that is also selected as a ligand is a methyl on His73. Separately selectable is an acetyl group at the N-terminus of each protein.

Question: What is the significance of the position of profilin in the complex with regard to the ability of the actin to:
(a) exchange ADP for ATP
(b) add to the plus end of an actin filament?


C N O S P Sr

Derivatives of the membrane lipid phosphatidylinositol are involved in signal cascades.

Signal-activated kinases convert phosphatidylinositol to PIP2 (phosphatidylinositol-4,5-bisphosphate).

Phospholipase C, activated by a signal cascade, catalyzes cleavage of PIP2 to yield the signal molecules diacylglycerol and inositol trisphosphate (IP3).

In addition to its role in generating the second messengers diacylglycerol and IP3, PIP2 formation and hydrolysis can affect the actin cytoskeleton.

For example, regulated formation and cleavage of PIP2 can affect profilin concentration, and hence actin polymerization, adjacent to the plasma membrane.

PIP2 binds profilin at the cytosolic surface of the plasma membrane. This prevents profilin-actin interaction. 

Signal-activated PIP2 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. 

Some actin severing and capping proteins also bind to PIP2, including gelsolin and cofilin. Filament severing can be a mechanism for increasing the number of plus ends to which actin can polymerize. Binding to PIP2 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.

Nucleation promoting factors that activate the Arp2/3 complex include proteins called WASP and Scar (also called WAVE). The genetic disease Wiskott-Aldrich Syndrome gave WASP its name.

WASP/Scar proteins have domains that bind and activate Arp2/3, plus domains that recognize and bind to various 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 PIP2 (phosphatidylinositol-4,5-bisphosphate). See A p. 947.

Some pathogens utilize a host cell's actin to move around in an infected cell and for transmission to other cells. They move by growth of actin tails. Diagram in A p. 1447.

A website, with movies showing actin-based movement of Listeria and Shigella within cells, is maintained by Dr. J. Theriot at Stanford School of Medicine.

Rho is a family of small GTP-binding proteins that regulate the actin cytoskeleton. Some members of the Rho family and the cytoskeletal structures they predominantly regulate are listed below.

In each case the active form of the Rho family protein has bound GTP.

Downregulation of Rho proteins involves GTPase activating proteins (GAPs) that facilitate GTP hydrolysis by Rho. In addition, guanine nucleotide dissociation inhibitors (GDIs) bind to Rho and prevent GDP/GTP exchange.

Activation of Rho proteins is by guanine nucleotide exchange proteins (GEFs) of the Dbl family that promote release of GDP with binding of GTP. The Dbl GEFs are in turn activated via signal cascades initiated via plasma membrane receptors such as cytokine receptors, growth factor receptors and cell adhesion receptors.

In addition to activating formins to promote actin filament growth, Rho-GTP promotes myosin-actin interactions essential for development and contraction of stress fibers, through its activation of ROCK (Rho Kinase).

ROCK phosphorylates (and inhibits dephosphorylation of) myosin II light chains. Light chain phosphorylation promotes interaction of myosin with actin filaments.

Other kinases that regulated formation or disassembly of focal adhesions include:

Proteins of the ERM family, including ezrin, radixin and moesin, provide regulated linkage of actin filaments to the plasma membrane in some cells. ERM proteins have actin-binding domains and domains that bind to cytosolic domains of plasma membrane integral proteins. They are regulated by signal-activated phosphorylation and by interaction with PIP2.

Calpains are Ca++-activated cysteine proteases that regulate cell adhesion. These intracellular proteases cleave constituents of stress fibers and focal adhesions during activation of cell motility. Proteins cleaved by calpains include actin-binding proteins such as a-actinin, filamin, talin and spectrin; subunits of plasma membrane integrins; and 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.

See also a website on actin & actin binding proteins, by the Maciver Lab at University of Edinburgh.

Copyright 1998-2007 by Joyce J. Diwan.   All rights reserved.

Additional material on Actin Cytoskeleton:
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