Nucleation of Actin Polymerization by Gelsolin

Abstract

The time-course of assembly of actin with gelsolin was measured by the fluorescence increase of a fluorescent label covalently linked to actin. The actin concentrations ranged from values far below the critical concentration to values above the critical concentration of the pointed ends of actin filaments. If the concentration of actin was in the range of the critical monomer concentration (0.64 pM), the time-course of the concentration of actin assembled with gelsolin revealed a sigmoidal shape. At higher actin concentrations the time-course of association of actin with gelsolin approximated an exponential curve. The measured time-courses of assembly were quantitatively interpreted by kinetic rate equations. A poor fit was obtained if two actin molecules were assumed to bind to gelsolin to form a 1 : 2 gelsolin-actin complex and subsequently further actin molecules were assumed to polymerize onto the 1 : 2 gelsolin-actin complex toward the pointed end. A considerably better agreement between calculated and measured time-courses was achieved if additional creation of actin filaments by fast fragmentation of newly formed actin filaments by not yet consumed gelsolin was assumed to occur. This suggests that both polymerization of actin onto gelsolin and fragmentation of actin filaments contribute to formation of new actin filaments by gelsolin. Furthermore it could be demonstrated that below the critical monomer concentration appreciable amounts of actin are incorporated into gelsolin-actin oligomers.

Power transduction of actin filaments ratcheting in vitro against a load

“…Critical concentration C crit and rate of monomer attachment k on and detachment k off at the pointed end in our salt conditions were derived elsewhere (21) from pyrene fluorescence assays (34). Results yielded C crit = 0:7 μM, k on = 0:12 μM −1 · s −1 , and k off = k on C crit = 0:084 s −1 .…”

The effect of two actin depolymerizing factors (ADF/cofilins) on actin filament turnover : pH sensitivity of F‐actin binding by human ADF, but not of Acanthamoeba actophorin

“…The dashed line shows for comparison the equivalent experiment using ADF from [7]. calculated from these experiments reflect both the dissociation rate constant, k Ϫ and the number concentration of ends N. The dissociation rate constant at the pointed ends of F-actin and gelsolin-capped filaments is approximately 0.3 s Ϫ1 [21,25]. Our preference for using exponential rate constants rather than the initial rates of the fluorescence change is that the former measure the whole disassembly reactions, at high rates of disassembly, the early phase is lost in the time of mixing the proteins and it is, therefore, unreliable to compare initial rates.…”

“…Depolymerization assays were usually carried out by adding actophorin or ADF to F-actin or capped filaments (containing 10Ϫ15% PI-actin) diluted to less than 200 nM (well below the critical concentration for gelsolin-capped filaments (~0.6 µM [25]) either in low salt (10 mM Tris/HCl, pH 8.0, 0.2 mM ATP, 0.2 mM CaCl 2 , 1Ϫ5 mM dithiothreitol and 1 mM NaN 3 ) or high salt (the same buffer containing also 1 mM MgCl 2 and 100 mM KCl). Dithiothreitol concentrations were maintained at 2Ϫ5 mM in ADF experiments to inhibit thiol oxidation and protein aggregation.…”

Phosphatidylinositol 4,5‐bisphosphate specifically stimulates PP60c‐src catalyzed phosphorylation of gelsolin and related actin‐binding proteins

“…Abbreviations: Cer, ceramide; DTT, dithiothreitol; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PI-3 kinase, phosphatidylinositol-3 kinase; PIP, phosphatidylinositol 4-monophosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PS, phosphatidylserine; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid growth of actin filaments by creating nucleation sites [15,17,18]. These activities are enhanced by Ca 2+ whereas polyphosphoinositides such as PIP2 and PIP are known to act as inhibitors, in particular of the severing activity (reviewed in [19]).…”