Time Course Of Polymerization Research Articles

Mutation of Actin Tyr-53 Alters the Conformations of the DNase I-binding Loop and the Nucleotide-binding Cleft

All but 11 of the 323 known actin sequences have Tyr at position 53, and the 11 exceptions have the conservative substitution Phe, which raises the following questions. What is the critical role(s) of Tyr-53, and, if it can be replaced by Phe, why has this happened so infrequently? We compared the properties of purified endogenous Dictyostelium actin and mutant constructs with Tyr-53 replaced by Phe, Ala, Glu, Trp, and Leu. The Y53F mutant did not differ significantly from endogenous actin in any of the properties assayed, but the Y53A and Y53E mutants differed substantially; affinity for DNase I was reduced, the rate of nucleotide exchange was increased, the critical concentration for polymerization was increased, filament elongation was inhibited, and polymerized actin was in the form of small oligomers and imperfect filaments. Growth and/or development of cells expressing these actin mutants were also inhibited. The Trp and Leu mutations had lesser but still significant effects on cell phenotype and the biochemical properties of the purified actins. We conclude that either Tyr or Phe is required to maintain the functional conformations of the DNase I-binding loop (D-loop) in both G- and F-actin, and that the conformation of the D-loop affects not only the properties that directly involve the D-loop (binding to DNase I and polymerization) but also allosterically modifies the conformation of the nucleotide-binding cleft, thus increasing the rate of nucleotide exchange. The apparent evolutionary "preference" for Tyr at position 53 may be the result of Tyr allowing dynamic modification of the D-loop conformation by phosphorylation (Baek, K., Liu, X., Ferron, F., Shu, S., Korn, E. D., and Dominguez, R. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 11748-11753) with effects similar, but not identical, to those of the Ala and Glu mutations.

Dynamic Imaging of Fibrin Network Formation Correlated with Other Measures of Polymerization.

The clotting time or gel point is commonly used in a variety of clinical assays, but little has been known about what it represents structurally because of the limitations of imaging technologies. The necessity of fixation and dehydration for electron microscopy means that only final clot structures, often distorted by preparation artifacts, can be observed, and there are severe limitations of image quality for conventional light microscopy. For similar reasons, we know almost nothing about the branching and lateral aggregation of fibrin that are essential for clot or thrombus stability. Turbidity, light scattering, or clot stiffness are useful to follow the time course of polymerization but reflect only overall clot properties. We visualized in real time fibrin network formation in the hydrated state, using deconvolution microscopy, which allows optical sectioning without the bleaching that accompanies confocal microscopy. Thus, the events during polymerization could be followed quantitatively over long periods of time. Videos will demonstrate the major observations. Individual mobile fibers were observed before the gel point. After gelation, an initial fibrin network, or scaffold, was seen, which evolved over time by addition of new fibers and elongation and branching of others. Furthermore, some fibers in the network moved chaotically for some time. A detailed, quantitative morphological analysis of network formation was carried out by superposition of images from different time points colorized to distinguish changes. We quantified network formation by the number of branch points, and longitudinal and lateral growth of fibers as a function of time. The distributions of fibers that reached a maximum of longitudinal growth and branch point formation both had maxima at the gel point but, surprisingly, some longitudinal growth continued and new branch points appeared after the gel point, requiring modification of existing models of fibrin polymerization. The cumulative percentage of fibers reaching their final length and the number of branch points attained maximum values at the time corresponding to that at which the turbidity reached approximately 60% of its maximum. Lateral growth reached a plateau at the same time as turbidity. Measurements of clot mechanical properties revealed that the clots achieved maximum stiffness and minimum plasticity well after branch point, as well as length changes and lateral growth of fibers, were completed. These results provide new information on the time sequence of events during fibrin network formation, which is important to understand both clotting and thrombosis and to allow modulation of clot properties.

Microscopic analysis of polymerization dynamics with individual actin filaments.

The polymerization-depolymerization dynamics of actin is a key process in a variety of cellular functions. Many spectroscopic studies have been performed in solution, but studies on single actin filaments have just begun. Here, we show that the time course of polymerization of individual filaments consists of a polymerization phase and a subsequent steady-state phase. During the steady-state phase, a treadmilling process of elongation at the barbed end and shortening at the pointed end occurs, in which both components of the process proceed at approximately the same rate. The time correlation of length fluctuation of the filaments in the steady-state phase showed that the polymerization-depolymerization dynamics follow a diffusion (stochastic) process, which cannot be explained by simple association and dissociation of monomers at both ends of the filaments.

H2O2-treated actin: assembly and polymer interactions with cross-linking proteins

During inflammation, hydrogen peroxide, produced by polymorphonuclear leukocytes, provokes cell death mainly by disarranging filamentous (polymerized) actin (F-actin). To show the molecular mechanism(s) by which hydrogen peroxide could alter actin dynamics, we analyzed the ability of H2O2-treated actin samples to polymerize as well as the suitability of actin polymers (from oxidized monomers) to interact with cross-linking proteins. H2O2-treated monomeric (globular) actin (G-actin) shows an altered time course of polymerization. The increase in the lag phase and the lowering in both the polymerization rate and the polymerization extent have been evidenced. Furthermore, steady-state actin polymers, from oxidized monomers, are more fragmented than control polymers. This seems to be ascribable to the enhanced fragility of oxidized filaments rather than to the increase in the nucleation activity, which markedly falls. These facts; along with the unsuitability of actin polymers from oxidized monomers to interact with both filamin and alpha-actinin, suggest that hydrogen peroxide influences actin dynamics mainly by changing the F-actin structure. H2O2, via the oxidation of actin thiols (in particular, the sulfhydryl group of Cys-374), likely alters the actin C-terminus, influencing both subunit/subunit interactions and the spatial structure of the binding sites for cross-linking proteins in F-actin. We suggest that most of the effects of hydrogen peroxide on actin could be explained in the light of the "structural connectivity," demonstrated previously in actin.

Direct evidence for ADP-Pi-F-actin as the major intermediate in ATP-actin polymerization. Rate of dissociation of Pi from actin filaments.

The sequence of reactions involved in the polymerization of ATP-actin and accompanying hydrolysis of ATP has been investigated by using a new glass-fiber filter assay. The assay allows the rapid separation of filaments from monomeric actin, and therefore the straightforward identification of the nucleotide bound to F-actin in the time course of polymerization, using double-labeled [gamma-32P,3H]ATP. The data bring a direct confirmation of the existence of the previously proposed ATP-F-actin intermediate in the time course of polymerization. Moreover, comparison of the hydrolyzed ATP (i.e., acid-labile [32P]Pi) and of 32P bound to F-actin provides direct evidence for the second intermediate ADP-Pi-F-actin in the polymerization process. This latter species is the major transient in the polymerization of ATP-actin, its lifetime being of the order of minutes.

Effects of thrombospondin on fibrin polymerization and structure.

Thrombospondin (TSP) is a trace protein in plasma but is released in high concentrations from alpha-granules of activated platelets during hemostasis. It binds to the platelet membrane and becomes incorporated into fibrin clots. A variety of approaches were taken to learn the effects of TSP on fibrin polymerization and structure. 125I-TSP and 125I-fibrinogen were used to study the effect of TSP concentration on the extent of TSP and fibrin incorporation. Turbidity at 600 nm was used to monitor the time course of polymerization. Wavelength dependence of the turbidity was used to calculate the mass to length ratio, fiber diameter, and fiber density of fibrin formed in the presence and absence of TSP. Morphologies of control and TSP-containing clots were examined by electron microscopy following critical point drying. The initial TSP concentration influenced the amount of TSP incorporated but did not alter the extent of fibrin polymerization. TSP, in a concentration-dependent manner, reduced the lag time to turbidity rise and caused formation of more numerous but thinner fibers. Except for their diameter, these fibers were identical to fibers of control fibrin in terms of density and morphology. It is proposed that TSP interacts with fibrin intermediates to accelerate fiber growth, perhaps by serving as a trifunctional branching unit during network formation. The properties of fibrin around aggregating platelets, therefore, may be influenced considerably by secreted TSP.

The rate of polymerization of rabbit skeletal muscle actin is enhanced by polyethylene glycol.

The effect of polyethylene glycol on the kinetics of actin polymerization was determined by monitoring the enhancement in the fluorescence of pyrenyl-labelled actin. The polymerization of actin at 15 mM KCl was in addition followed by viscometry and light scattering. All three methods showed that the overall rate of polymerization of actin increased 3-4-fold when the concentration of polyethylene glycol was increased from 0 to 6% (ww-1). A further increase in polyethylene glycol concentration to 10% (ww-1) caused a relatively small contribution to the increase in the rate of polymerization. The enhancement of the overall rate of polymerization by polyethylene glycol was also reflected in a significant decrease in the lag time observed when the time course of polymerization was followed by viscometry and light scattering. The steady-state value of fluorescence enhancement and critical concentration of actin were also influenced by polyethylene glycol and the results showed that the extent of polymerization was increased by an increase in the concentration of polyethylene glycol in solution. The effect of polyethylene glycol on both rate and extent of polymerization persisted at physiological salt concentration (150 mM KCl, 2 mM MgCl2). Since the rate of elongation was affected only to a small extent by polyethylene glycol, we propose that its main effect is on nucleation.

Polymerization of human transcortin in plasma

Using radial immunodiffusion and antiserum raised against purified transcortin, polymers of native transcortin have been identified in plasma. They are characterized by multiple precipitin bands, the size of which diminishes on dilution, consistent with reversibility of this form of polymer. Spectrophotometric scans have been used to study the time course of polymerization in purified transcortin, in which dilution reversibility can be demonstrated also, and in which disaggregation may be produced by addition of sodium dodecyl sulphate and dithiothreitol, but apparently not by cortisol.

Polymerization of actin and actin-like systems: evaluation of the time course of polymerization in relation to the mechanism.

The time course of protein polymerization of the nucleation--elongation type is examined by using a general computer-simulation solution. For a simple nucleation--elongation scheme, it is shown that the half-time of polymerization is not necessarily a good measure of the nucleus size as has been previously suggested [Oosawa, F., & Kasai, M. (1962) J. Mol. Biol. 4, 10-21] since, depending on the mechanism, the apparent nucleus size, measured by a ratio of half-times at two actin concentrations, may be either larger or smaller than the real size. Steady-state equations developed by Wegner and Engel [Wegner, A., & Engel, J. (1975) Biophys. Chem. 3, 215-225] present a good description of the time course of polymerization although they are somewhat inflexible with regard to allowing for different mechanisms. Some of the assumptions implicit in the development of these equations are discussed in terms of the effect of changing individual rate constants or dissociation constants on the time course of polymerization. In addition, these steady-state equations have been expanded to include the consequences of a reversible first-order conformational change prior to polymerization. It is shown that a conformational change as a prerequisite to polymerization lengthens the lag time of polymerization and, depending on the conditions, may slow the rate of polymerization. The question of fragmentation and of reannealling is examined, and it is noted that simple relationships to describe these processes may not be possible.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymerization of actin: mechanism of the Mg2+-induced process at pH 8 and 20 degrees C.

A detailed mechanism that fully accounts for the Mg2+-induced polymerization of actin in the presence or absence of Ca2+ at 20 degrees C and pH 8 is presented. In the absence of Ca2+, the mechanism of the Mg2+-induced polymerization is as follows: Mg2+ binds to a metal-binding site on G-actin and induces a conformational change, which is required for eventual polymerization. The overall dissociation constant for this binding is about 30 microM. This actin species then binds a second molecule of Mg2+ (Kd = 5 mM), which yields a species capable of polymerization. Dimer formation from this monomeric species is quite unfavorable, but trimer formation from dimer and monomer is much more favorable. The trimer may then elongate to give filaments. Ca2+, when present, binds at the same site as the tightly bound Mg2+ and must be displaced by Mg2+ before the conformational change can occur. The rate and dissociation constants for tight binding of Ca2+ and Mg2+ and for the conformational change are consistent with those observed previously by using a fluorescently labeled G-actin. With the mechanism proposed, it is possible to fit the full time course of polymerization over a wide range of actin concentrations, Mg2+ concentrations, and Ca2+ concentrations.

Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization.

The fluorescence of pyrene-labelled actin is much higher after polymerization. We have characterized in detail the polymerization properties of pyrene actin and report that native and pyrene actin are identical using the following criteria: (1) the time course of polymerization; (2) the elongation rate constants; (3) the intrinsic viscosity; and (4) the critical concentration. Native and pyrene actin copolymerize. Fluorescence of polymerized pyrene actin is 7-10 times higher than monomer. The fluorescent signal is proportional to polymer weight concentration and is insensitive to filament length distribution. Bleaching can be minimized by appropriate filters to allow continuous monitoring of signal. Measurements do not influence polymerization kinetics. This establishes that pyrene actin fluorescence is a valid assay for actin polymerization that is more sensitive than any other current assay.

Kinetic analysis of actin assembly suggests that tropomyosin inhibits spontaneous fragmentation of actin filaments

The time-course of actin assembly was measured in the absence and in the presence of tropomyosin. The polymerization was followed by the fluorescence enhancement of a 7-chloro-4-nitrobenzeno-2-oxa-1,3-diazole label attached to actin molecules or by light-scattering. The kinetic curves measured in the absence and in the presence of tropomyosin revealed characteristic differences. Tropomyosin was found to retard actin polymerization and to cause the final constant actin monomer concentration to be reached slowly. In the absence of tropomyosin, the final constant actin monomer concentration was approached considerably faster. The time-course of polymerization was interpreted quantitatively in terms of inhibition of actin filament fragmentation by tropomyosin molecules bound along the filaments. Within the limits of this model, actin monomers are consumed slowly in the presence of tropomyosin because the creation of new filament ends by spontaneous fragmentation is inhibited by tropomyosin.

Deoxyribonucleic acid-dependent ribonucleic acid polymerase of Caulobacter crescentus.

Deoxyribonucleic acid-dependent ribonucleic acid (RNA) polymerase (EC 2.7.7.6) was purified from the dimorphic bacterium Caulobacter crescentus at three stages in development. Enzyme from pure populations of stalked cells, as well as populations enriched in swarmer and predivisional cells, appeared identical in subunit structure and template requirements. The molecular weights of the enzyme subunits were 165,000, 155,000, 101,000, and 44,000, respectively. By analogy with RNA polymerase from other bacterial sources, they are considered to be components of the C. crescentus holoenzyme, beta', beta, sigma, and alpha, respectively. The C. crescentus enzyme appeared similar to the Pseudomonas aeruginosa enzyme and unlike the Escherichia coli enzyme with respect to subunit molecular weights and failure to separate into core and sigma components upon phosphocellulose chromatography. In addition, the effects of ionic strength on the time course of polymerization varied both with the sources of bacterial polymerase and bacteriophage DNA.