Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis.

Way, Michael; Gooch, John; Pope, Brian; Weeds, Alan G.

doi:10.1083/jcb.109.2.593

Cited by 199 publications

(194 citation statements)

References 49 publications

(94 reference statements)

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“…S1). Weak F-actin depolymerizing activity exhibited by G2-G6 at higher molar ratios is in accordance with the earlier reports (10 (Fig. 4C).…”

Section: Regulation Of F-actin Depolymerizing Activity Of Truncated Gsupporting

confidence: 93%

Global Shapes of F-actin Depolymerization-competent Minimal Gelsolins

Peddada¹,

Sagar²,

Rathore³

et al. 2013

Journal of Biological Chemistry

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Background: Shape-function studies are necessary to design better therapeutic alternatives of the plasma gelsolin. Results: N-terminal fragment 30 -161 is the smallest segment with F-actin depolymerization potential, and G1-G3 can function independent of Ca 2ϩ ions or low pH. Conclusion: The g2-g3 linker plays a role in imparting pH/Ca 2ϩ insensitivity to G1-G3. Significance: We provide the first evidence that g2-g3 linker regulates mobility of the G1 domain.

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“…S1). Weak F-actin depolymerizing activity exhibited by G2-G6 at higher molar ratios is in accordance with the earlier reports (10 (Fig. 4C).…”

Section: Regulation Of F-actin Depolymerizing Activity Of Truncated Gsupporting

confidence: 93%

Global Shapes of F-actin Depolymerization-competent Minimal Gelsolins

Peddada¹,

Sagar²,

Rathore³

et al. 2013

Journal of Biological Chemistry

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“…For biochemical analysis, 10 ml of the same buffers for each 100-mm dish were used. In some experiments, VPMs were preincubated for 3 min at 0°C in HCB containing 4 M fusion protein corresponding to the full-length gelsolin (Way et al, 1989), a generous gift from Dr. Michael Way (European Molecular Biology Laboratory, Heidelberg, Germany). After incubation at 37°C, VPMs were immediately processed for immunofluorescence or for biochemical analysis.…”

Section: Cell-free Assaymentioning

confidence: 99%

A Cell-free System to Study Regulation of Focal Adhesions and of the Connected Actin Cytoskeleton

Cattelino

Albertinazzi

Bossi

et al. 1999

MBoC

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Assembly and modulation of focal adhesions during dynamic adhesive processes are poorly understood. We describe here the use of ventral plasma membranes from adherent fibroblasts to explore mechanisms regulating integrin distribution and function in a system that preserves the integration of these receptors into the plasma membrane. We find that partial disruption of the cellular organization responsible for the maintenance of organized adhesive sites allows modulation of integrin distribution by divalent cations. High Ca 2ϩ concentrations induce quasi-reversible diffusion of ␤1 integrins out of focal adhesions, whereas low Ca 2ϩ concentrations induce irreversible recruitment of ␤1 receptors along extracellular matrix fibrils, as shown by immunofluorescence and electron microscopy. Both effects are independent from the presence of actin stress fibers in this system. Experiments with cells expressing truncated ␤1 receptors show that the cytoplasmic portion of ␤1 is required for low Ca 2ϩ -induced recruitment of the receptors to matrix fibrils. Analysis with function-modulating antibodies indicates that divalent cation-mediated receptor distribution within the membrane correlates with changes in the functional state of the receptors. Moreover, reconstitution experiments show that purified ␣-actinin colocalizes and redistributes with ␤1 receptors on ventral plasma membranes depleted of actin, implicating binding of ␣-actinin to the receptors. Finally, we found that recruitment of exogenous actin is specifically restricted to focal adhesions under conditions in which new actin polymerization is inhibited. Our data show that the described system can be exploited to investigate the mechanisms of integrin function in an experimental setup that permits receptor redistribution. The possibility to uncouple, under cell-free conditions, events involved in focal adhesion and actin cytoskeleton assembly should facilitate the comprehension of the underlying molecular mechanisms.

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“…The isoforins containing the essential light chains A1 or A2 were separated by chromatography on DEAE-cellulose. Human cytoplasmic gelsolin and gelsolin segment 1 were expressed in transformed Escherichia coli and prepared from inclusion bodies as described previously [22]. DNase I is product of Worthington Corp. and was further purified by chromatography on hydroxyapatite according to Mannherz et al 1231.…”

Section: Protein Preparationsmentioning

confidence: 99%

“…This effect could be due to re-equilibration of ADP-actin back to the monomeric pool, due to the lower critical concentration of ADP-actin or to a further increase in the number of filament ends due to the severing activity of gelsolin [35]. In support of the latter possibility is the observation that gelsolin segment 2-6, which possesses nucleating but no severing activity [22,371, only induced a monophasic actin polymerization from the Tp4 complex under identical conditions (data not shown). It is noteworthy that the non-muscle actin failed to show this fluorescence reversal.…”

Section: Polymerization Of Actin From Actin :Tp4 Complex Induced By Hmentioning

confidence: 99%

Induction of the polymerization of actin from the actin:thymosin β4 complex by phalloidin, skeletal myosin subfragment 1, chicken intestinal myosin I and free ends of filamentous actin

Ballweber

Hannappel

Niggemeyer

et al. 1994

European Journal of Biochemistry

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Thymosin /34 is able to form 1 : 1 complexes with monomeric (G) actin, thereby stabilizing the intracellular pool of unpolymerized actin. We have searched for factors that are able to induce the polymerization of actin from the actin : thymosin p4 complex. Phalloidin, subfragment 1 isolated from rabbit skeletal muscle myosin and chicken intestinal myosin I are demonstrated to be able to polymerize the actin from this complex in the presence of 1 mM MgCl,. Polymerization of actin was verified by the DNase I inhibition assay, by cosedimentation and from the fluorescence increase of pyrene-labelled actin. Actin filaments formed under the influence of subfragment 1 or phalloidin were visualized under the electron microscope after negative staining. Polymerization of skeletal muscle actin from the complex with thymosin p4 by phalloidin is accompanied by the hydrolysis of the actin-bound ATP to ADP. Polymerization was also induced by sonicated F-actin which possessed a high concentration of free filament ends. F-actin was severed by 0.01 M human cytoplasmic gelsolin, which is known to possess blocked + ends. Free, slowly growing -ends were unable to induce polymerization of actin from the thymosin p4 complex. However, when gelsolin on its own or in complex with two actin molecules was added to actin:thymosin p4 under nucleating conditions, it was found to be able to promote actin repolymerization provided that its concentration was close to the dissociation constant (Kd) of actin:thymosin p4. This Kd was found to be 0.4 yM in the presence of 1 mM MgC1, and the absence of KC1 and, thus, close to the critical concentration of actin polymerization under these conditions. The source of actin did not influence its polymerization from the thymosin p4 complex ; rabbit skeletal muscle actin and porcine brain actin were polymerized with equal efficiency from their complexes with thymosin /34 by both phalloidin and myosin subfragment 1. Skeletal muscle, but not cytoplasmic actin, was found to be also polymerized in the presence of increased CaC1, concentrations to values above 1 mM.Actin is present at a high concentration in virtually all types of eukaryotic cells. About 50% of the intracellular actin is stabilized in the monomeric form, although the ionic conditions of the cytoplasm favour its complete polymerization to F-actin. There is now ample evidence that approximately 50% of the intracellular actin is stabilized in the monomeric form in complexes with sequestering factors. This unpolymerized actin is able to enter the polymeric pool after an appropriate intracellular or extracellular signal. In 1990, Safer et al. [l] identified a peptide of approximately 5 kDa in platelets that preferentially binds to G-actin, even in the presence of high salt, thus stabilizing the monomeric pool of G-actin. This protein was found to be identical to thymosin p4 (Tp4) [2], a peptide originally believed to be a thymic hormone [3] and able to modulate the immune responsiveness of peripheral lymphocytes [4, 51. However, the unex-

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Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis.

Abstract: Abstract. Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli.

Cited by 199 publications

References 49 publications

Global Shapes of F-actin Depolymerization-competent Minimal Gelsolins

Global Shapes of F-actin Depolymerization-competent Minimal Gelsolins

A Cell-free System to Study Regulation of Focal Adhesions and of the Connected Actin Cytoskeleton

Induction of the polymerization of actin from the actin:thymosin β4 complex by phalloidin, skeletal myosin subfragment 1, chicken intestinal myosin I and free ends of filamentous actin

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