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Regulation of the F-actin severing activity of gelsolin by Ca2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca2+ binding sites with affinities of 2.5 x 10(7) M-1 and 1.5 x 10(5) M-1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 microM free Ca2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at approximately 0.4 microM free Ca2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca2+ concentrations. The data suggest that binding of Ca2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca2+ binding event is a committed step that results in a Ca2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow.

Gelsolin is a Ca2+-regulated actin-binding protein that can sever, cap, and nucleate growth from the pointed ends of actin filaments. In this study we have measured the binding of the amino-terminal half of gelsolin, G1-3, to pyrene-labeled F-actin as a function of Ca2+ concentration. The rate of binding is shown to be dependent on micromolar concentrations of Ca2+. Independent experiments demonstrate that conformational changes in G1-3 are induced by micromolar concentrations of Ca2+. Titrations of pyrene-F-actin with G1-3 and gelsolin show that the quenching of pyrene fluorescence is identical in extent and stoichiometry for both G1-3 and gelsolin. In contrast, severing of F-actin by G1-3 is found to be much less efficient than is severing by gelsolin. In experiments in which F-actin severing is quantitatively measured, the filament number is found to be proportional to the 1.35 power of the G1-3 concentration. This deviation from linearity may be explained by cooperativity; the binding of two G1-3 molecules in close proximity may lead to cooperative severing of the polymer, thus increasing the severing efficiency. This model is supported by experiments that show that the efficiency of G1-3 severing of F-actin increases with increasing G1-3:F-actin ratios. Extrapolating from these results, we conclude that G4-6, the carboxyl-terminal half of gelsolin, has an active role in the severing of F-actin by intact gelsolin. Whereas F-actin severing by G1-3 is enhanced by cooperative binding of two separate G1-3 molecules, cooperativity is inherent to intact gelsolin because the cooperative partners are covalently linked.

SynopsisA structural transition is reported to occur in aqueous sols of agarose, an electrically uncharged biostructural polysaccharide. The transition has no measurable effect on size dispersity on the shape of the solute polysaccharide as observed by precision photon correlation spectroscopy. It originates a low-angle pattern of scattered light similar to that which monitors phase separations in polymer blends. Thus, it must be due to some extent to spatially modulated polymer clustering, typical of spinodal decomposition. In the interval of temperatures studied, it precedes very distinctly in time the thermoreversible sol-gel transition, which is known to be promoted a t higher concentrations. It also anticipates to an appreciable extent the spatial density modulation observed in the gel. Although reported here for the first time, a spinodal decomposition of the sol that precedes and possibly triggers the processes leading to gelation does not come unexpectedly in terms of site-bond correlated-percolation theory. In general, this occurrence raises the question as t o whether the spontaneous onset of regions of higher and lower polymer concentration (spinodal separation) may be regarded as a novel path for biomolecuiar interactions and the self-assembly of order in biomolecular systems.

The delay or lag phase at the onset of polymerization of actin by neutral salt is generally attributed to an actin nucleation reaction. However, when nucleation is circumvented by the use of phalloidin-stabilized nuclei, a lag phase persists when Ca2+-containing actin is polymerized with MgCl2. Pretreatment of actin with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and/or Mg2+ shortens or eliminates this lag phase, suggesting that exchange of the actin-bound divalent cation occurs during this nucleation-independent lag phase. Measurement of the actin-bound cation initially and after brief incubation with EGTA/Mg2+ directly verifies that Mg2+ has replaced Ca2+ as the actin-bound cation, producing a highly polymerizable Mg2+-actin species. Bound-cation exchange prolongs the lag phase in actin polymerization and probably explains what has been termed the monomer activation step in actin polymerization.

No abstract

Gel formation and spatial structure is an important area of study in polymer physics and in macromolecular and cellular biophysics. Agarose has a sufficiently complex gelation mechanism to make it an interesting prototype for many other gelling systems, including those involved in amyloid fibrillogenesis. Static (over a scattering vector range of 0.1-30 microm(-1)) and dynamic light scattering and rheology methods were used to follow the gelation kinetics of agarose at 0.5% in water or in the presence of 25 mM NaCl and quenched to temperatures of 20-43 degrees C. Light scattering results on gelling samples are fully described by a fractal aggregate model with four physically meaningful parameters. In all cases aggregates, with fractal dimensions at or near 3, form more rapidly and are smaller in characteristic size at lower quench temperatures. A region three to four times larger than the aggregate becomes depleted of agarose as the gelation proceeds. Below about 30 degrees C the aggregation process freezes spatial ordering rapidly, resulting in fragile macroscopic gels as determined by rheology. Salt effects are seen to be minimal and not important in the fundamental aggregation mechanism.

Measurement of the diffusion coefficient of G-actin using fluorescence photobleaching recovery reveals that a hydrodynamically larger species is formed in Mg2+-containing buffers but not in Ca2+-containing buffers. In buffer A (2 mM Tris-HCl, 0.2 mM CaCl2, 0.2 mM ATP, and 0.5 mM 2-mercaptoethanol, pH 8.0) and other Ca2+-containing buffers, the data indicate a single species with a diffusion coefficient of (7.98 ± 0.17) X 10™7 cm2/s. In Mg2+-containing buffers the average diffusion coefficient was (5.52 ± 0.06) X 10~7 cm2/s, and there was evidence of more than one component. The distinction between these buffer systems was reversible by dialysis. The conjecture that the putative oligomeric species may be active in promoting actin assembly was confirmed in two independent experiments. Assembly of actin in the presence of these species produced shorter filaments, as would be expected if they act to increase the number of nucleation sites. In addition, trace quantities of these species added to buffer A actin had the ability to accelerate the kinetics of actin assembly. It is concluded that G-actin below the critical concentration forms oligomeric species in the presence of 50-200 µ Mg2+ and that these species either are assembly nuclei or at least are more readily converted to assembly nuclei than is monomeric actin. e capacity of actin for reversible self-assembly is believed to be an indispensable element of its role in cytoplasmic mo-