Severing of F-Actin by the Amino-Terminal Half of Gelsolin Suggests Internal Cooperativity in Gelsolin

Selden, Lynn A.; Kinosian, Henry J.; Newman, Jay; Lincoln, Bryan; Hurwitz, Charles; Gershman, Lewis C.; Estes, James E.

doi:10.1016/s0006-3495(98)77750-1

Cited by 57 publications

(52 citation statements)

References 28 publications

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“…Both Kid fragments moved smoothly and stably for several micrometers, showing that the second microtubule-binding site does not inhibit the motility of Kid. Some of these movements may be induced by several Kid molecules because it has been reported that the plural gelsolin molecules bound to the same actin filament (29). Therefore, it is unclear from our assay whether Kid is a processive motor or not.…”

Section: Resultsmentioning

confidence: 95%

The Second Microtubule-binding Site of Monomeric Kid Enhances the Microtubule Affinity

Shiroguchi

Ohsugi

Edamatsu

et al. 2003

Journal of Biological Chemistry

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Chromokinesin Kid (kinesin-like DNA-binding protein) localizes on spindles and chromosomes and has important roles in generating polar ejection force on microtubules in the metaphase. To understand these functions of Kid at the molecular level, we investigated molecular properties of Kid, its oligomeric state, interaction with microtubules, and physiological activity in vitro. Kid expressed in mammalian cells, as well as Kid expressed in Escherichia coli, was found to be monomeric. However, Kid cross-linked microtubules in an ATP-sensitive manner, suggesting that Kid has a second microtubule-binding site in addition to its motor domain. This was ascertained by binding of Kid fragments lacking the motor domain to microtubules. The interaction of the second microtubule-binding site was weak in a nucleotide-insensitive manner. K mMT of the ATPase activity of Kid was lower than that of the fragments lacking the second microtubule-binding site. Moreover, the velocity of Kid movement in vitro was not affected by the second microtubule-binding site, which is consistent with the weak binding of this site to microtubules. The second microtubule-binding site would be important to enhance the affinity to microtubules for the monomeric motor, Kid. Because the amino acid sequence of this region is highly conserved among species, it seems to have essential roles for the functions of Kid in vivo.

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Section: Resultsmentioning

confidence: 95%

The Second Microtubule-binding Site of Monomeric Kid Enhances the Microtubule Affinity

Shiroguchi

Ohsugi

Edamatsu

et al. 2003

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

“…A truncated ⑀-amino rhodamine-labeled peptide (1-25 rhod-t␤ 4 peptide) was produced synthetically with a sequence that corresponds to the N-terminal 25 residues of thymosin ␤ 4 , SDKPDMAEIEKFDKSKLKK-TETQEK-rhodamine. Human recombinant gelsolin was truncated to the N-terminal 406 residues (identified here as gelsolin segment 1-3) prior to expression so as to eliminate the dependence of F-actin binding on calcium (27).Anisotropy Assay-A standard curve showing the fluorescence anisotropy of a rhodamine-labeled thymosin ␤ 4 peptide (0.1 M) as a function of Mg 2ϩ -actin concentration in F buffer (5.0 mM Tris-HCl, 40 mM KCl, 2.0 mM MgCl 2 , 0.2 mM ATP, 0.2 mM dithiothreitol, 0.1 mM CaCl 2 , 0.125 mM EGTA, and 0.01% sodium azide, pH 7.8) was generated with vertically excited polarized light at 546 nm in an L or T format steady state fluorimeter. The KCl concentration was 8.0 mM for the experiments employing unlabeled thymosin ␤ 4 , and in this low salt buffer the standard curve as shown in Fig.…”

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

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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...

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“…5 In spite of the absence of strict regulation of the actin filament-severing ability of G1-G3, calcium ion binding to this fragment does influence its structure and activity, as demonstrated by a reduced hydrodynamic volume, diminished proteolytic susceptibility, and increased tryptophan fluorescence. 9,10 Isolated G1 experiences a three-orders of magnitude increase in affinity for actin in the presence of calcium ions, and the Ca 2C occupying its type II site is reported to be locked into the G1-actin structure, inaccessible to chelation by EGTA. 11 Furthermore, transient binding of Ca 2C to G2 is proposed to facilitate the activation process.…”

Section: Introductionmentioning

confidence: 99%

Calcium Ion Exchange in Crystalline Gelsolin

Chumnarnsilpa

Loonchanta

Xue

et al. 2006

Journal of Molecular Biology

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Gelsolin is a calcium and pH-sensitive modulator of actin filament length. Here, we use X-ray crystallography to examine the extraction and exchange of calcium ions from their binding sites in different crystalline forms of the activated N and C-terminal halves of gelsolin, G1-G3 and G4-G6, respectively. We demonstrate that the combination of calcium and low pH activating conditions do not induce conformational changes in G4-G6 beyond those elicited by calcium alone. EGTA is able to remove calcium ions bound to the type I and type II metal ion-binding sites in G4-G6. Constrained by crystal contacts and stabilized by interdomain interaction surfaces, the gross structure of calcium-depleted G4-G6 remains that of the activated form. However, high-resolution details of changes in the ionbinding sites may represent the initial steps toward restoration of the arrangement of domains found in the calcium-free inactive form of gelsolin in solution. Furthermore, bathing crystals with the trivalent calcium ion mimic, Tb 3C , results in anomalous scattering data that permit unequivocal localization of terbium ions in each of the proposed type I and type II ionbinding sites of both halves of gelsolin. In contrast to predictions based on solution studies, we find that no calcium ion is immune to exchange.

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Severing of F-Actin by the Amino-Terminal Half of Gelsolin Suggests Internal Cooperativity in Gelsolin

Cited by 57 publications

References 28 publications

The Second Microtubule-binding Site of Monomeric Kid Enhances the Microtubule Affinity

The Second Microtubule-binding Site of Monomeric Kid Enhances the Microtubule Affinity

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

Calcium Ion Exchange in Crystalline Gelsolin

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