Ca2+ Regulation of Gelsolin Activity: Binding and Severing of F-actin

Abstract

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.

Visualizing the Ca 2+ -dependent activation of gelsolin by using synchrotron footprinting

“…State 1 corresponds to the ''Ca ϩ2 -free'' form, state 2 is the intermediate observed in these studies that involves some unlatching of the structure, and state 3 is the Ca 2ϩ -saturated fully activated form. The transition between states 1 and 2 occurs at submicromolar concentrations, which is in a good agreement with the previous biochemical studies (1,9,15,19,21), and is accompanied by the binding of multiple Ca 2ϩ ions. The transition between states 2 and 3 is mediated by occupancy of lower-affinity binding sites and is accompanied by the binding of two or three additional Ca 2ϩ ions (see Description The data of Fig.…”

“…The intracellular Ca 2ϩ concentration is tightly regulated in the range of 10 Ϫ7 to 10 Ϫ5 M, whereas millimolar concentrations are common in plasma. A number of biophysical approaches, including fluorescence (9,(14)(15)(16)(17)(18), equilibrium dialysis (19,20), dynamic light scattering (21), and x-ray crystallography (4,6,10), have been used to examine the binding of Ca 2ϩ to gelsolin and the Ca 2ϩ -induced structural reorganization associated with its actin-binding and -severing activity (22)(23)(24). The occupancy of very-high-affinity sites (K d Ͻ 0.1 M), likely within S5 or S6, appears to induce a significant conformational reorganization, including the possible release of the S6 latch that sequesters the F-actin-binding site present in S2 (21).…”

Transcriptional profiles associated with aging and middle age-onset caloric restriction in mouse hearts

“…Tropomyosin stabilizes the actin cytoskeleton, whereas plastins and fascins organize filamentous actins into bundles (45)(46)(47). Cofilin enhances actin filament turnover (48), and gelsolin regulates actin assembly (49,50). These findings suggest major structural remodeling in response to CR.…”

Dendritic spine actin cytoskeleton in autism spectrum disorder

“…Gelsolin severs F-actin filaments by breaking the noncovalent bonds between actin monomers, and remains bound to the barbed ends of the filaments, inhibiting their extension 84,85 ( Figure 1). Dendritic spines are motile structures that contain high concentrations of filamentous actin, and based on a fluorescence recovery after photobleaching (FRAP) experiment in hippocampal neurons, the great majority (around 85%) of the F-actin in the spines is dynamic 86 .…”