Interaction of ADP-ribosylated actin with actin binding proteins.

Actin ADP-ribosylated at Arg177 was previously shown not to polymerise after increasing the ionic strength, but to cap the barbed ends of filaments. Here we confirm that the polymerisation of ADP-ribosylated actin is inhibited, however, under specific conditions the modified actin copolymerises with native actin, indicating that its ability to take part in normal subunit interactions within filaments is not fully eliminated. We also show that ADP-ribosylated actin forms antiparallel but not parallel dimers: the former are not able to form filaments. ADP-ribosylated actin interacts with deoxyribonuclease I, vitamin D binding protein, thymosin beta(4), cofilin and gelsolin segment 1 like native actin. Interaction with myosin subfragment 1 revealed that the potential of the modified actin to aggregate into oligomers or short filaments is not fully eliminated.     

Polyamine-induced actin polymerization.

Muscle actin has been found to polymerize reversibly upon addition of low concentrations of polyamines. This polymerization, studied by centrifugation, has shown a linear relationship between the actin polymerization yield and the chain length of the polyamine. Among the biological polyamines tested, spermidine and spermine are the most efficient. The polymerization of actin can also be induced by the corresponding mono or diguanidine derivatives of these polyamines but monoamines or amino acids are inactive at the same concentration. The transformation of actin from a globular to a fibrous from upon addition of spermidine is also demonstrated by the changes in the near-ultraviolet circular dichoroic spectrum of this protein. Moreover, the polyamine-induced F -actin exhibits the same properties as the salt-induced F -actin: it strongly activates the Mg2+ -ATPase of myosin, its specific viscosity is enhanced to the same extent and electron micrographs show homogeneous thin filaments.     

Birefringence of actin.

The total strain birefringence of F-actin isolated from chicken gizzards was measured as a function of elongation in thin transparent films. Each film held at a certain elongation in a jig was allowed to swell in a penetrating but nondissolving liquid. Seven liquids with different refractive indices were employed. The thickness of the film in each swelling liquid was obtained once equilibrium was established. At each elongation, from 0 to 16%, a Wiener curve was obtained. The minima of the Wiener curves yielded the intrinsic birefringence of F-actin as a function of elongation. The intrinsic birefringence increases with elongation up to 16%, above which the thin films break. The form birefringence at a set refractive index also increases with elongation. The implication of the strain birefringence of F-actin is discussed as it affects the optical properties, mainly light scattering, of tissues such as the fiber cells of lens of the eye.     

Ascaris suum actin: properties and similarities to rabbit actin.

Unlike other enzymes of the aromatic multienzyme system, chorismate synthase and the aromatic complex of $\text{Neurospora crassa}$ were found to bind to a column of cellulose phosphate and to elute at a relatively high concentration of phosphate (～ 0.2 $\text{M}$). The fact that other enzymes with similar ionic properties failed to bind to phosphocellulose suggests that the binding of the former two enzyme systems is due to a specific affinity for phosphate. This conclusion is not only supported by the fact that these same enzymes did not bind to a column of carboxymethyl cellulose, but also is consistent with the nature of the catalytic reactions of the enzymes. Both the shikimate kinase enzyme of the aromatic complex and chorismate synthase would be expected to have active sites which accomodate a phosphate moiety. We anticipate that other enzymes which involve phospho-substrates will also be amendable to this procedure.     

Kinetics of the cooperative association of actin to actin filaments.

The cooperative formation of actin filaments from monomers was followed by light scattering and electron microscopy. The results are well described by a simple model mechanism in which the growth and destruction of filaments occurs by stepwise addition or dissociation of protomers. All steps except the dimerisation step are assumed to have identical rate constants. These were found to be 5 X 10(3) M-1 - sec-1 and 3 X 10(-2) sec-1 for the association and dissociation, respectively (at pH 7.5 and in the presence of 10(-3) M calcium chloride). The equilibrium constant of elongation as obtained from the critical concentration is 1.7 X 10(5) M-1. The corresponding equilibrium constant of dimerisation is about 10 million times smaller (cooperativity parameter sigma = 2 X 10(-7)). This makes the nucleation extremely difficult and cooperativity very high. A best fit of the model to the experimental data is achieved when the destruction of a dimer is much faster than the addition of a third protomer (fast monomer- dimer pre-equilibrium). The size of the nucleus from which propagation becomes faster than dissociation is 3.     

The polymerization of actin. III. Aggregates of nonfilamentous actin and its associated proteins: a storage form of actin

When echinoderm sperm are treated with the detergent Triton X-100 at pH 6.4 in 10 mM phosphate buffer, the membranes are solubilized, but the actin which is located in the periacrosomal region remains as a phase-dense cup. These cups can be isolated free from the flagella and chromatin and can be solubilized by increasing the pH to 8.0 and by changing the ionic strength and type of buffer used. Since the actin does not exist in the "F" state in unreacted sperm, and since the actin remains as a unit that does not diffuse away, it must be present in the mature sperm in a bound or storage state. The actin is, in fact, associated with a pair of proteins whose mol wt are 250,000 and 230,000. When the isolated cups are digested with trypsin, these high molecular weight proteins are digested, thereby liberating the actin. The actin will polymerize if heavy meromyosin or subfragment 1 is added to a preparation of isolated cups. Evidence is presented that this pair of high molecular weight proteins is similar in molecular weight and properties to erythrocyte spectrin. Attempts at transforming the storage form of actin in the cup into filaments were only moderately successful. The best conditions for filament formation involve incubating the cup in ATP and divalent salts. Careful examination of these cups reveals that the actin polymerized preferentially on either end of oriented filaments that already exist in the cup, indicating that self-nucleation is inefficacious. I conclude that the actin can exist in the storage form by its association with spectrin-like molecules and that the actin in this state polymerizes preferentially onto existing filaments.     

Water in actin polymerization.

We have addressed the question whether water is part of the G- to F-actin polymerization reaction. Under osmotic stress, the critical concentration for G-Ca-ATP actin was reduced for six different osmolytes. These results are interpreted as showing that reducing water activity favored the polymerized state. The magnitude of the effect correlated, then saturated, with increasing MW of the osmolyte and suggested that up to 10-12 fewer water molecules were associated with actin when it polymerized. By contrast, osmotic effects were insignificant for Mg-ATP actin. The nucleotide binding site of the Mg conformation is more closed than the Ca and more closely resembles the closed actin conformation in the polymerized state. These results suggest that the water may come from the cleft of the nucleotide binding site.     

Polymerization of Acanthamoeba actin. Kinetics, thermodynamics, and co-polymerization with muscle actin.

The kinetics and thermodynamics for the polymerization of purified Acanthamoeba actin were studied and compared to muscle actin. Polymerization was qualitatively similar for the two actins with a rate-limiting nucleation step followed by rapid polymer extension. Polymerization occurred only above a threshold critical concentration which varied with polymerization conditions for each actin. In the presence of 2 mM MgCl2, nucleation of both actins was rapid and their critical concentrations were similarly low and not detectably dependent on temperature. In 0.1 M KCl, the rates of nucleation of both actins were much slower than when Mg2+ was present and were significantly different from each other. Also, under these conditions, the critical concentrations of Acanthamoeba and muscle actin were significantly different and both varied markedly with temperature. These quantitative differences between the two actins could be attributed to differences in both their enthalpies and entropies of polymerization, Acanthamoeba actin having the more positive deltaH and delta S. Co-polymerization of the two actins was also demonstrated. Overall, however, there were no qualitative differences between Acanthamoeba and muscle actin that would suggest a unique role for the monomer-polymer equilibrium of cytoplasmic actin in cell motility.     

Postsynaptic actin and neuronal plasticity.

In the adult brain, actin is concentrated in dendritic spines where it can produce rapid changes in their shape. Through various synaptic junction proteins, this postsynaptic actin is linked to neurotransmitter receptors, influencing their function and, in turn, being influenced by them. Thus, the actin cytoskeleton is emerging as a key mediator between signal transmission and anatomical plasticity at excitatory synapses.     

Ca2+ control of actin gelation. Interaction of gelsolin with actin filaments and regulation of actin gelation

We elucidated the mechanism by which gelsolin, a Ca2+-dependent regulatory protein from lung macrophages, controls the network structure of actin filaments. In the presence of micromolar Ca2+, gelsolin bound Ca2+. The Ca2+-gelsolin complex reduced the apparent viscosity and flow birefringence of F-actin and the lengths of actin filaments viewed in the electron microscope. However, concentrations of gelsolin causing these alterations did not effect proportionate changes in the turbidity of actin filament solutions or in the quantity of nonsedimentable actin as determined by a radioassay. From these findings, we conclude that gelsolin shortens actin filaments without net depolymerization. Such an effect on the distribution of actin filament lengths led to the prediction that low concentrations of gelsolin would increase the critical concentration of actin-binding protein required for incipient gelation of actin filaments in the presence of Ca2+, providing an efficient mechanism for controlling actin network structure. We verified the prediction experimentally, and we estimated that the Ca2+-gelsolin complex effectively breaks the bond between actin monomers in filaments with a stoichiometry of 1:1. The effect of Ca2+-gelsolin complex on actin solation was rapid, independent of temperature between 0 degrees and 37 degrees C, and reversed by reducing the free Ca2+ concentration.     

The actin content of fibroblasts.

Cultures of chick skin fibroblasts were dissolved in solutions of sodium dodecyl sulphate, and their entire protein content was examined by gel electrophoresis. The most abundant species migrated in the same position as muscle actin. It gave a similar pattern of iodinated peptides after reaction with radioactive sodium iodide and digestion with proteinases, and contained comparable amounts of Nt-methylhistidine. Its amount was estimated by quantitative densitometry of stained gels with bovine serum albumin as an internal standard, and by radioactive assay of cultures that had been grown in the presence of [35S]methionine. The values obtained ranged from 7 to 14% of the total cellular protein, with an average of 8.5%. A protein band in the position of muscle myosin was also present and accounted for about 2.5% of the total protein. Both this and the actin band increased in relative amount with the age of the cultures.     

Immobilization of actin and myosin.

Using glutaric dialdehyde, the muscle proteins myosin, actin, actomyosin and heavy meromyosin subfragment-1 (S-1) have been immobilized on capron fibers. The ATPase activity of myosin and its capability to interact with actin have been preserved whereas the ATPase activity of its subfragment decreased significnatly. Immobilization on capron fibers changes the pH dependence of the ATPase activity of myosin and of S-1 shifting the maximum towards the acid zone (pH 5.5) and increases the thermal stability of the enzyme. Calcium ions produce a stimulatory effect on ATPase; Mg2+ions yield no effect on myosin and S-1 but enhance the activity in the case of immobilized actomyosin though to a lesser degree than the ions of Ca2+. Immobilized actin retains its ability to form actomyosin complex.     

pH dependence of actin self-assembly.

Fluorescence enhancement and fluorescence photobleaching recovery have been utilized to examine actin self-assembly over the pH range 6.6-8.0. The kinetics of assembly are faster and the critical concentrations are lower at lower pH. Filament diffusion coefficients are not a function of pH, indicating that average filament lengths are not pH dependent. Although critical actin concentrations are a sensitive function of the concentrations of various cations in the medium, the relative pH dependences of critical concentrations are similar for all combinations of cations employed. The pH dependence of actin self-assembly is sufficiently great that it should be taken into account when comparing data from different reports and when relating in vitro measurements to cytoplasmic mechanisms.     

Putting a new twist on actin: ADF/cofilins modulate actin dynamics.

The actin-depolymerizing factor (ADF)/cofilins are a family of essential actin regulatory proteins, ubiquitous among eukaryotes, that enhance the turnover of actin by regulating the rate constants of polymerization and depolymerization at filament ends, changing the twist of the filament and severing actin filaments. Genetic and cell-biological studies have shown that an ADF/cofilin is required to drive the high turnover of the actin cytoskeleton observed in vivo. The activity of ADF/cofilin is regulated by a variety of mechanisms, including specific phosphorylation and dephosphorylation. This review addresses aspects of ADF/cofilin structure, dynamics, regulation and function.