Rapid and efficient purification of actin from nonmuscle sources

Schafer, Dorothy A.; Jennings, Phillip B.; Cooper, John A.

doi:10.1002/(sici)1097-0169(1998)39:2<166::aid-cm7>3.0.co;2-4

Cited by 39 publications

(26 citation statements)

References 32 publications

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“…Cellular actin was extracted as per Schafer et al (32). Cells were lysed in actin extraction buffer (1.0 M Tris⅐HCl pH 7, 1 mM DTT, 0.6 M KCl, 0.5 mM MgCl 2, 0.5 mM ATP, 0.2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 4% Triton X-100, and 2% Tween 20), and then lysates were ultracentrifuged at 110,000 g for 60 min at 4°C to remove DNA and particulates.…”

Section: Immunoblot Analysis Of Actinmentioning

confidence: 99%

Peroxynitrite mediates TNF-α-induced endothelial barrier dysfunction and nitration of actin

Neumann¹,

Gertzberg²,

Vaughan³

et al. 2006

American Journal of Physiology-Lung Cellular and Molecular Phys

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Section: Immunoblot Analysis Of Actinmentioning

confidence: 99%

Peroxynitrite mediates TNF-α-induced endothelial barrier dysfunction and nitration of actin

Neumann¹,

Gertzberg²,

Vaughan³

et al. 2006

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…Native poppy profilin was purified as described (Clarke et al, 1998), and native poppy actin was isolated by DNase I chromatography (Schafer et al, 1998) followed by electroelution from SDS-PAGE gels. Standard curves of purified profilin or actin from poppy pollen were prepared in the range of 0 to 5 ng.…”

Section: Quantification Of Total Actin and Total Profilinmentioning

confidence: 99%

Signal-Mediated Depolymerization of Actin in Pollen during the Self-Incompatibility Response

et al. 2002

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Signal perception and the integration of signals into networks that effect cellular changes is essential for all cells. The self-incompatibility (SI) response in field poppy pollen triggers a Ca 2 ؉ -dependent signaling cascade that results in the inhibition of incompatible pollen. SI also stimulates dramatic alterations in the actin cytoskeleton. By measuring the amount of filamentous (F-) actin in pollen before and during the SI response, we demonstrate that SI stimulates a rapid and large reduction in F-actin level that is sustained for at least 1 h. This represents quantitative evidence for s timulus-mediated depolymerization of F-actin in plant cells by a defined biological stimulus. Surprisingly, there are remarkably few examples of sustained reductions in F-actin levels stimulated by a biologically relevant ligand. Actin depolymerization also was achieved in pollen by treatments that increase cytosolic free Ca 2 ؉ artificially, providing evidence that actin is a target for the Ca 2 ؉ signals triggered by the SI response. By determining the cellular concentrations and binding constants for native profilin from poppy pollen, we show that profilin has Ca 2 ؉ -dependent monomeric actinsequestering activity. Although profilin is likely to contribute to stimulus-mediated actin depolymerization, our data suggest a role for additional actin binding proteins. We propose that Ca 2 ؉ -mediated depolymerization of F-actin may be a mechanism whereby SI-induced tip growth inhibition is achieved.

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“…Sixty milligrams of deoxyribonuclease (DNase I, Sigma, St. Louis, type IV) was coupled to 2.5 g of cyanogen bromide-activated Sepharose 4B (Pharmacia Biotech, Piscataway, NJ) according to Schafer et al (1998).…”

Section: Preparation Of Deoxyribonuclease I Affinity Chromatography Cmentioning

confidence: 99%

Identification and Characterization of a Ca2+-Dependent Actin Filament-Severing Protein from Lily Pollen

et al. 2004

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It is well known that a tip-focused intracellular Ca 21 gradient and the meshwork of short actin filaments at the tip region are necessary for pollen tube growth. However, little is known about the connections between the two factors. Here, a novel Ca 21 -dependent actin-binding protein with molecular mass of 41 kD from lily (Lilium davidii) pollen (LdABP41) was isolated and purified with DNase I chromatography. Our purification procedure yielded about 0.6 mg of LdABP41 with .98% purity from 10 g of lily pollen. At least two isoforms with isoelectric points of 5.8 and 6.0 were detected on two-dimensional gels. The results of N-terminal sequencing and mass-spectrometry analysis of LdABP41 showed that both isoforms shared substantial similarity with trumpet lily (Lilium longiflorum) villin and other members of the gelsolin superfamily. Negative-stained electron microscope images showed that LdABP41 severed in vitro-polymerized lily pollen F-actin into short actin filaments in a Ca 21 -sensitive manner. Microinjection of the anti-LdABP41 antibody into germinated lily pollen demonstrated that the protein was required for pollen tube growth. The results of immunolocalization of the protein showed that it existed in the cytoplasm of the pollen tube, especially focused in the tip region. Our results suggest that LdABP41 belongs to the gelsolin superfamily and may play an important role in controlling actin organization in the pollen tube tip by responding to the oscillatory, tip-focused Ca 21 gradient.Pollen tube growth is a key process in the sexual reproduction of higher plants. It is highly polarized and requires both spatial and temporal coordination of many cellular functions, including ion fluxes, cytoskeleton organization and dynamics, vesicular trafficking, exocytosis, endocytosis, and cell wall synthesis (for review, see Taylor and Hepler, 1997;Franklin-Tong, 1999). It is widely accepted that the actin cytoskeleton plays a major role in modulation of pollen tube growth. Actin filaments, together with myosin, are a crucial element to support intracellular trafficking of organelles and secretory vesicles along actin cables that are oriented axially throughout the shank of elongating pollen tubes (Cai et al., 1997;Vidali and Hepler, 2000;Hepler et al., 2001). Although the presence and exact distribution of F-actin in the pollen tube apex has been somewhat controversial, accumulating evidence indicates that a highly dynamic array of actin filaments in the tip region may play a primary role in polarized growth. Early studies using chemically fixed cells and fluorescent-phalloidin staining indicate that there is a dense actin network at the extreme apex of pollen tubes (Pierson, 1988;Derksen et al., 1995). However, investigations with rapid freezing, freeze substitution microscopy techniques suggest that the extreme apex of pollen tubes contains little F-actin, but only occasional sparse or fine actin filaments at the extreme apex (Doris and Steer, 1996). Results obtained by microinjection of trumpet lily (Lilium lo...

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Rapid and efficient purification of actin from nonmuscle sources

Cited by 39 publications

References 32 publications

Peroxynitrite mediates TNF-α-induced endothelial barrier dysfunction and nitration of actin

Peroxynitrite mediates TNF-α-induced endothelial barrier dysfunction and nitration of actin

Signal-Mediated Depolymerization of Actin in Pollen during the Self-Incompatibility Response

Identification and Characterization of a Ca2+-Dependent Actin Filament-Severing Protein from Lily Pollen

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