Direct measurement of critical concentrations and assembly rate constants at the two ends of an actin filament

Bonder, Edward M.; Fishkind, Douglas J.; Mooseker, Mark S.

doi:10.1016/0092-8674(83)90382-3

Cited by 131 publications

(94 citation statements)

References 41 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2B). The barbed end elongation rate constant, extrapolated to 100% unlabeled actin, was 8 M Ϫ1 ⅐s Ϫ1 , similar to values determined by electron microscopy (6,8). The elongation rate constant extrapolated to zero at 0.22 M total actin, which corresponded to 0.13 M unlabeled actin (Fig.…”

Section: Resultssupporting

confidence: 53%

“…We observed actin filament elongation and branch formation mediated by Arp2͞3 complex in real time. Direct measurements of filament growth confirmed the rate constants measured by electron microscopy (6,8) and established that rhodamine actin is a kinetically inactive tracer for imaging. In the presence of activated Arp2͞3 complex, growing actin filaments formed branches at random sites along their sides, rather than preferentially from their barbed ends, providing further support for the side-binding model for dendritic nucleation (13,17,18).…”

mentioning

confidence: 49%

“…Bulk biochemical methods for studying actin polymerization have been available for more than a half-century (5) and were initially useful for determining the conditions required for shifting the monomer-polymer equilibrium. Later electron microscopic studies allowed filament elongation rates to be determined directly by measuring their lengths after elongating for known periods (6)(7)(8). More recent studies using fluorescently labeled phalloidin to visualize filaments have allowed direct visualization of filament severing (9)(10)(11) and have shown the products of annealing (12) and branch formation mediated by Arp2͞3 complex (13,14).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy

Amann

Pollard

2001

Proc. Natl. Acad. Sci. U.S.A.

246

211

View full text Add to dashboard Cite

Existing methods for studying actin filament dynamics have allowed analysis only of bulk samples or individual filaments after treatment with the drug phalloidin, which perturbs filament dynamics. Total internal reflection fluorescence microscopy with rhodamine-labeled actin allowed us to observe polymerization in real time, without phalloidin. Direct measurements of filament growth confirmed the rate constants measured by electron microscopy and established that rhodamine actin is a kinetically inactive tracer for imaging. In the presence of activated Arp2͞3 complex, growing actin filaments form branches at random sites along their sides, rather than preferentially from their barbed ends.

show abstract

Section: Resultssupporting

confidence: 53%

mentioning

confidence: 49%

mentioning

confidence: 99%

See 1 more Smart Citation

Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy

Amann

Pollard

2001

Proc. Natl. Acad. Sci. U.S.A.

246

211

View full text Add to dashboard Cite

show abstract

“…The measured values are in good agreement with the rate constants determined by other laboratories. Based on direct observation by electron microscopy of the elongation of actin filaments a + twofold greater rate constant of elongation of the barbed ends was reported [47,48]. Rate constants determined by nucleated polymerization onto actin timers tended to be smaller [51].…”

Section: Discussionmentioning

confidence: 99%

Rate constants and equilibrium constants for binding of the gelsolin‐actin complex to the barbed ends of actin filaments in the presence and absence of calcium

Selve

Wegner

1986

European Journal of Biochemistry

View full text Add to dashboard Cite

The equilibrium constant for binding of the gelsolin‐actin complex to the barbed ends of actin filaments was measured by the depolymerizing effect of the gelsolin‐actin complex on actin filaments. When the gelsolin‐actin complex blocks monomer consumption at the lengthening barbed ends of treadmilling actin filaments, monomers continue to be produced at the shortening pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at the uncapped barbed ends. By using this effect the equilibrium constant for binding was determined to be about 1.5 × 1010 M−1 in excess EGTA over total calcium (experimental conditions: 1 mM MgCl2, 100 mM KCl, pH 7.5, 37°C). In the presence of Ca2+ the equilibrium constant was found to be in the range of or above 1011 M−1. The rate constant of binding of the gelsolin‐actin complex to the barbed ends was measured by inhibition of elongation of actin filaments. Nucleation of new filaments by the gelsolin‐actin complex towards the pointed ends was prevented by keeping the monomer concentration below the critical monomer concentration of the pointed ends where the barbed ends of treadmilling actin filaments elongate and the pointed ends shorten. The gelsolin‐actin complex was found to bind fourfold faster to the barbed ends in the presence of Ca2+ (10 × 106 M−1 s−1) than in excess EGTA (2.5 × 106 M−1 s−1). Dissociation of the gelsolin‐actin complex from the barbed ends can be calculated to be rather slow. In excess EGTA the rate constant of dissociation is about 1.7 × 10−4 s−1. In the presence of Ca2+ this dissociation rate constant is in the range of or below 10−4 s−1.

show abstract

“…Measurement of these rate constants using a method such as electron microscopy can be used to indirectly evaluate A c , assuming the applicability of the theory (13,14). However, the participation of actin bound to monomer-sequestering proteins in filament growth, such as occurs with profilin-actin (15,16), greatly complicates this analysis, because such addition alters the observed rate constants but, in theory, can occur with (9) or without (17) a change in A c .…”

mentioning

confidence: 99%

Effects of Profilin and Thymosin β4 on the Critical Concentration of Actin Demonstrated in Vitro and in Cell Extracts with a Novel Direct Assay

Yarmola

Bubb

2004

Journal of Biological Chemistry

View full text Add to dashboard Cite

The free actin concentration at steady state, A c , is a variable that determines how actin regulatory proteins influence the extent of actin polymerization. We describe a novel method employing fluorescence anisotropy to directly measure A c in any sample after the addition of a trace amount of labeled thymosin ␤ 4 or thymosin ␤ 4 peptide. Using this assay, we confirm earlier theoretical work on the helical polymerization of actin and confirm the effects of actin filament-stabilizing drugs and capping proteins on A c , thereby validating the assay. We also confirm a controversial prior observation that profilin lowers the critical concentration of Mg 2؉ -actin. A general mechanism is proposed to explain this effect, and the first quantitative dose-response curve for the effect of profilin on A c facilitates its evaluation. This mechanism also predicts the effect of profilin on critical concentration in the presence of the limited amount of capping protein, which is the condition often found in cells, and the effect of profilin on critical concentration in cell extracts is demonstrated for the first time. Additionally, nonlinear effects of thymosin ␤ 4 on the steady state amount of F-actin are explained by the observed changes in A c . This assay has potential in vivo applications that complement those demonstrated in vitro.Total cellular actin is equal to the sum of the concentration of free monomer (the critical concentration, A c ), the concentration of unpolymerized actin sequestered by various actin-binding proteins, and the concentration of actin polymer or F-actin. Changes in A c induced by actin regulatory proteins influence actin polymer dynamics by an amplification mechanism that results in large changes in the amount of sequestered, unpolymerized actin. Quantitative evaluation of models of cytoskeletal function requires accurate knowledge of the value of A c . Unfortunately, A c has not yet been measured in cells and has only been estimated by very indirect methods in cell extracts (1). In vitro methods have been developed that allow for the measurement of F-actin using birefringence (2), viscosity (3), light scattering (4), centrifugation (5), binding of labeled phalloidin (6), or the fluorescence of pyrenyl-labeled actin (7). Other methods such as the DNase I binding assay yield the sum of A c and an indeterminate fraction of sequestered actin monomer (8). Clever uses of combinations of data have in some cases allowed investigators to subtract F-actin content from total actin and then fractionate the contributions of A c and monomer sequestration (9, 10), but the results have proven controversial (11,12).Based on theoretical considerations, the critical concentration is expected to be equal to the ratio of the rate constants of association and dissociation of actin subunits from actin filaments. Measurement of these rate constants using a method such as electron microscopy can be used to indirectly evaluate A c , assuming the applicability of the theory (13,14). However, the participation of actin b...

show abstract

Direct measurement of critical concentrations and assembly rate constants at the two ends of an actin filament

Cited by 131 publications

References 41 publications

Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy

Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy

Rate constants and equilibrium constants for binding of the gelsolin‐actin complex to the barbed ends of actin filaments in the presence and absence of calcium

Effects of Profilin and Thymosin β4 on the Critical Concentration of Actin Demonstrated in Vitro and in Cell Extracts with a Novel Direct Assay

Contact Info

Product

Resources

About