12-fold difference between the critical monomer concentrations of the two ends of actin filaments in physiological salt conditions.

Abstract

We determined the critical monomer concentrations at which association and dissociation reactions are balanced at the two ends of actin filaments. For measurement of the critical concentration of the pointed end, interference with the high dynamics of the barbed end was excluded by capping the barbed ends with an actin filament capping protein isolated from bovine brain. The critical concentration of the pointed end (1.5 /AM) was found to be 12-to 15-fold higher than the critical concentration of the barbed end (0.10-0.12 jAM) at a temperature of 37°C and physiological salt concentrations (100 mM KCI/1-2 mM MgCI2/ 0.3 mM EGTA or 0.2 mM CaC12, pH 7.5).Actin filaments have been shown in vitro to lengthen at the barbed end and to shorten simultaneously at the pointed end under steady-state conditions (1)(2)(3). This treadmilling process is driven by a continuous ATP hydrolysis occurring during the association of actin monomers with a filament end (1, 4). At the barbed end of actin filaments, a lower critical monomer concentration is required to balance association and dissociation reactions as compared with the pointed end. The critical concentration of the pointed end could not be measured directly because association and dissociation at the barbed end occurs much faster than at the pointed end. Therefore, events occurring at the pointed end of filaments are difficult to observe without interfering with the dynamics of the barbed end (5, 6).In this study this problem was overcome by capping the barbed filament end with an actin filament capping protein from bovine brain (7,8). Capped filaments labeled with 7-chloro-4-nitrobenz-2-oxa,1,3-diazole were mixed with various concentrations of unlabeled monomeric actin. The monomer concentration range in which filaments can release subunits from the pointed ends was determined by a fluorescence change accompanying the monomer-polymer transition of labeled actin molecules. The critical concentration of the barbed end was measured by the reverse assay. Unlabeled filaments were mixed with various concentrations of labeled actin monomers. The monomer concentration range in which the barbed ends consume monomers was again determined by a fluorescence change. MATERIAL AND METHODSPreparation of the Proteins. Actin was prepared from rabbit skeletal muscle as described (9), including gel filtration on a Bio-Gel P-150 column. Part of the protein was modified with N-ethylmaleimide at cysteine-374 and subsequently with a fluorescent label (7-chloro-4-nitrobenz-2-oxa-1,3-diazole) at lysine-373 (10). Actin filament capping protein was extracted from bovine brain and purified by several ammonium sulfate fractionations and column chromatography steps-according to a published method (8). The concentration of actin was determined photometrically at 290 nm by using an extinction coefficient of 24,900 M1 cm-1 (1). The concentration of labeled actin was measured by the method of Lowry et al. (11), and the concentration of capping protein was determined as described by Bradford (12).F...

Does actin bind to the ends of thin filaments in skeletal muscle?

“…This was also the case in the absence of ATP (data not shown). These findings can be ascribed to the fact that the critical concentration at the pointed end is higher than that at the barbed end (14,36). We inferred that the critical concentrations at pointed ends and barbed ends in solution are, respectively, higher and lower than 0.1 mg/ml, so that the apparent critical concentration obtained in solution becomes 0. l mg/ml.…”

“…Furthermore, the binding constant of actin monomer to the pointed end is smaller than that to the barbed end (14,23,36). In this connection, it would be interesting to examine the conditions under which the binding of actin to the pointed end of thin filaments in myofibrils occurs, as well as to measure both the association rate and the association constant and to compare them with those obtained in the in vitro experiments.…”

Reconstitution of a prokaryotic minus end-tracking system using TubRC centromeric complexes and tubulin-like protein TubZ filaments

“…This behavior is in contrast to microtubules and ParM filaments, which display dynamic instability (21,22). In TubZ filaments, subunits are lost and added each at one specialized end only, similar to filamentous actin, which treadmills (23).…”

FtsZ filament capping by MciZ, a developmental regulator of bacterial division

“…These cappers usually bind to the growing (plus) end of filaments and restrict exchange of subunits to the minus end. Because the minus end has a higher Cc than the plus end, blocking the plus end leads to extensive depolymerization of filaments, until the free monomer concentration becomes equal to the minus-end Cc (39,40). Such depolymerization induced by eukaryotic capping proteins is dependent on, and a hallmark of, treadmilling (41).…”