Proteins that cross-link actin filaments can either form bundles of parallel filaments or isotropic networks of individual filaments. We have found that mixtures of actin filaments with alpha-actinin purified from either Acanthamoeba castellanii or chicken smooth muscle can form bundles or isotropic networks depending on their concentration. Low concentrations of alpha-actinin and actin filaments form networks indistinguishable in electron micrographs from gels of actin alone. Higher concentrations of alpha-actinin and actin filaments form bundles. The threshold for bundling depends on the affinity of the alpha-actinin for actin. The complex of Acanthamoeba alpha-actinin with actin filaments has a Kd of 4.7 microM and a bundling threshold of 0.1 microM; chicken smooth muscle has a Kd of 0.6 microM and a bundling threshold of 1 microM. The physical properties of isotropic networks of cross-linked actin filaments are very different from a gel of bundles: the network behaves like a solid because each actin filament is part of a single structure that encompasses all the filaments. Bundles of filaments behave more like a very viscous fluid because each bundle, while very long and stiff, can slip past other bundles. We have developed a computer model that predicts the bundling threshold based on four variables: the length of the actin filaments, the affinity of the alpha-actinin for actin, and the concentrations of actin and alpha-actinin.
To evaluate the contributions of cross-linker dynamics and polymer deformation to the frequency-dependent stiffness of actin filament gels, we compared the rheological properties of actin gels with three types of cross-linkers: a weak one, Acanthamoeba alpha-actinin (dissociation rate constant 5.2 s-1, association rate constant 1.1 x 10(6) M-1 s-1); a strong one, chicken smooth muscle alpha-actinin (dissociation rate constant 0.66 s-1, association rate constant 1.20 x 10(6) M-1 s-1); and an extremely strong one, biotin/avidin (dissociation rate constant approximately zero). The biotin/avidin cross-linked gel, whose behavior is determined by polymer bending alone, behaves like a solid and shows no frequency dependence. The amoeba alpha-actinin cross-linked gel behaves like a viscoelastic fluid, and the frequency dependence of the stiffness can be explained by a mathematical model for dynamically cross-linked gels. The stiffness of the chicken alpha-actinin cross-linked gel is independent of frequency, and has viscoelastic properties intermediate between the two. The two alpha-actinins have similar association rate constants for binding to actin filaments, consistent with a diffusion-limited reaction. Rigid cross-links make the gel stiff, but make it elastic without the ability to deform permanently. Dynamically cross-linked actin filaments should allow the cell to react passively to various outside forces without any sort of signaling. Slower, signal-mediated pathways, such as severing filaments or changing the affinity of cross-linkers, could alter the nature of these passive reactions.
We present evidence for a new mechanism by which two major actin monomer binding proteins, thymosin beta 4 and profilin, may control the rate and the extent of actin polymerization in cells. Both proteins bind actin monomers transiently with a stoichiometry of 1:1. When bound to actin, thymosin beta 4 strongly inhibits the exchange of the nucleotide bound to actin by blocking its dissociation, while profilin catalytically promotes nucleotide exchange. Because both proteins exchange rapidly between actin molecules, low concentrations of profilin can overcome the inhibitory effects of high concentrations of thymosin beta 4 on the nucleotide exchange. These reactions may allow variations in profilin concentration (which may be regulated by membrane polyphosphoinositide metabolism) to control the ratio of ATP-actin to ADP-actin. Because ATP-actin subunits polymerize more readily than ADP-actin subunits, this ratio may play a key regulatory role in the assembly of cellular actin structures, particularly under circumstances of rapid filament turnover.
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