Actin-sequestering

Because of its abundance in the cytosol and ability to bind ATP-G-actin (but not F-actin), thymosin (34 is considered to be the main actin-sequestering protein in cells. A small protein (5000 MW), thymosin binds ATP-G-actin in a 1 1 complex. The binding of thymosin 4 blocks the ATP-binding site in G-actin, thereby preventing its polymerization. In platelets, the concentration of thymosin B4 is 0.55 mM, approximately twice the concentration of unpolymerized actin (0.22 mM). At these concentrations, approximately 70 percent of the monomeric actin in a platelet should be sequestered by thymosin (34. 

Swinholide A, isolated from the marine sponge Theonella swinhoei, sequesters actin dimers and induces their formation. One molecule of swinholide A binds to one dimer. In addition, swinholide A can sever F-actin by binding to the neighbouring protomers. Increased depolymerization of F-actin has also been reported. 

Proteins that bind to actin monomers and inhibit polymerization are designated as profilins (12—15 kD) (Sun et al., 1995). In addition to functioning as an actin-monomer-sequestering protein, profilin binds at least three other... 

The marine macrolides latrunculin A and the less potent variation latrunculin B (5-25 pg/mL, 60 minutes) bind to actin and disrupt the cytoskeleton at low concentrations (90,91). Their mechanism of action includes binding to and sequestering actin monomers, resulting in filament depolymerization (89). 

Note that if a capping protein binds to monomeric actin, the capping protein will also be a monomer-sequestering agent. A good example of such behavior is profilin. See also ABM-1 ABM-2 Sequences inActin-Based Motors Actin-Based Bacterial Motility Actin Assembly Kinetics... 

Sequestering Proteins Inhibit Assembly by Increasing the Fraction of Actin Present in Monomer Forms... 

Sequestered monomers bound to the sequestering protein cannot bind to an actin hlament, and only free actin can participate in elongation. Thus,... 

In the absence of a sequestering protein, actin polymerization will proceed to equilibrium or it will reach a steady-state extent of polymerization, at which point d[Apoiy]/dt = 0. [A] 00 is the critical concentration, equal to kJk+. In the presence of a sequestering protein, [Atotai] = [Afree] + [Apoiy] + [AX], and after polymerization reaches equilibrium, the unpolymerized actin concentration [A]oo will equal [AX ] + [Afree]. Hence,... 

In the fibroblast, and other cells, the concentration of monomeric actin can be as high as 200fiM and represent up to 50% of the total actin in the cell. This contrasts sharply with the critical concentration of ATP-actin in vitro which is typically 1 fxM or less. Thymosin (Mr = 5,000) is one protein that is responsible for sequestering monomeric actin in these cells. A large pool of monomeric actin provides the opportunity to rapidly assemble an actin-based cytoskeletal network while leaving preexisting actin-based networks intact. 

Recently, Kosmin has shown that bistramide A is responsible for severing actin filaments and covalently modifying actin.183 These studies indicated that, while the spiroketal and amide units of the natural product induced rapid disassembly of F-actin in vitro, the enone subunit of bistramide A was able to initiate covalent modification of actin in vitro and in live cells. These results indicate that, while PKC5 may not be the primary target of the natural product, it plays a dual role by binding to and severing F-actin and covalently sequestering... 

Lipid raft domains of plasma membranes are enriched in cholesterol and sphingolipids. As a consequence, compounds that extract or sequester cholesterol, such as fS-cyclodextrins, nystatin, and filipin, can block selectively endocytosis of cholera toxin, GPI-linked proteins, and other receptors that associate with lipid rafts and caveolae. However, cholesterol is also critical for CME, secretion of proteins, and the actin network. Therefore, conditions designed to affect selectively raft-mediated endocytosis by perturbing cholesterol levels must be carefully controlled to avoid disrupting other mechanisms of endocytosis (40). 

Goode BL, Drubin DG, X ppalainen P. Regulation of the cortical actin cytoskeleton in budding yeast by twinfilin, a ubiquitous actin monomer-sequestering protein. J Cell Biol 1998 142(3) 723-733. 

Inhibition of Actin Assembly by Thymosin (34 Calculations based on the Q of G-actin (0.1 (jlM), a typical cytosolic total actin concentration (0.5 mM), and the ionic conditions of the cell indicate that nearly all cellular actin should exist as filaments there should be very little G-actin. Actual measurements, however, show that as much as 40 percent of actin in an animal cell is unpolymerized. What keeps the cellular concentration of G-actin above its Q The most likely explanation is that cytosolic proteins sequester actin, holding it in a form that is unable to polymerize. 

In a simple equilibrium, an increase in the cytosolic concentration of thymosin P4 would Increase the concentration of sequestered actin subunits and correspondingly decrease F-actln, because actin filaments are in equilibrium with actin monomers. This effect of thymosin P4 on the cellular F-actin level has been experimentally demonstrated in live cells. 

Promotion of Actin Assembly by Profilin Another cytosolic protein, profilin (15,000 MW), also binds ATP-actin monomers in a stable 1 1 complex. At most, profilin can buffer 20 percent of the unpolymerized actin in cells, a level too low for it to act as an effective sequestering protein. Rather than sequestering actin monomers, the main function of profilin probably is to promote the assembly of actin filaments in cells. It appears to do so by several mechanisms. 

G-actin. Actin subunits (pink) complexed with thymosin p4 (purple) dissociate (O)and add to the end of a filament (B). In the filament, ATP is hydrolyzed to ADR the ADP-associated subunit eventually dissociates from the opposite end of the filament (B), the ADP-G-actin forms a complex with profilin (green) (B), and ATP exchanges with ADP to form ATP-G-actin (B). Profilin delivers actin monomers to the (-t) end of actin filaments (B) or thymosin (J4 sequesters the ATP-G-actin into a polymerization ready pool of subunits (H). 

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