Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods.

Knowles, Gisele; McCulloch, C

doi:10.1177/40.10.1527379

Cited by 84 publications

(62 citation statements)

References 21 publications

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“…Phalloidins are known to interact with high affinity with filamentous F-actin but not with monomeric G-actin, and can therefore be used to quantitate the amount of polymerized actin in fixed cells. 25) As shown in Fig. 4, in uninfected cells, well-organized actin filaments were clear.…”

Section: Fig 1 Effects Of Different Inhibitors On the Internalizatimentioning

confidence: 66%

High Mobility Group Nucleosomal Binding Domain 2 Protein Protects Bladder Epithelial Cells from Klebsiella pneumoniae Invasion

Cao

Fan

et al. 2011

Biological & Pharmaceutical Bulletin

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Due to the predominance of multiple-antibiotic-resistant Klebsiella pneumoniae strains, the search for new approaches for the prevention of K. pneumoniae infections is now under intensive investigation. The objective of the present study was to investigate the effects of high mobility group nucleosomal binding domain 2 (HMGN2) protein, which acts on the bladder epithelial cells T 24, on the invasion of K. pneumoniae 03183 and explore its possible mechanisms. Pretreatment with HMGN2 significantly reduced K. pneumoniae 03183 uptake by T 24 cells. In T 24 cells, there were no detectable cytotoxic effects of HMGN2 at any concentration between 32 and 256 m mg/ml after 2 h incubation. HMGN2 exhibited no appreciable antibacterial activity against K. pneumoniae 03183. Fluorescence microscopy and flow cytometry analysis revealed that HMGN2 blocked K. pneumoniae 03183-induced actin polymerization. K. pneumoniae 03183-induced phosphorylation of extracellular signal-regulated kinase (ERK) and cofilin were prevented by pretreatment with HMGN2. These results indicated that pretreatment with HMGN2 inhibited cofilin phosphorylation and then induced actin disruption which may block ERK phosphorylation. These changes led to inhibition of K. pneumoniae 03183 invasion of T 24 bladder epithelial cells.Key words high mobility group nucleosomal binding domain 2; actin; Klebsiella pneumoniae; extracellular signal-regulated kinase; cofilin Biol. Pharm. Bull. 34(7) 1065-1071 (2011) © 2011 Pharmaceutical Society of Japan * To whom correspondence should be addressed. e-mail: huangpanxiaoxiao@sina.com tein Assay Kit (Pierce, U.S.A.) and the purity was confirmed by Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with a primary antibody mouse anti-His monoclonal antibody (Roche, U.K.).Invasion Assay For the invasion assay, T 24 cells were seeded at 5ϫ10 4 cells per well in 24-well plates. Cells were preincubated with substances (rHMGN2 or cytochalasin B or latrunculin B or Y27632 or PD98059) for 2 h prior to bacterial infection in order to study the cell structures and processes involved in internalization. The monolayers were washed twice with PBS to remove any substances prior to infection with K. pneumoniae 03183. In each well of a 24-well cell culture cluster, about 5ϫ10 6 mid-log-phase K. pneumoniae 03183 (OD 600 ϭ0.4-0.6) were added to epithelial cells per well. Invasion was allowed to occur for 2 h at 37°C in 5% CO 2 . Before a second 2 h incubation (kill) period under the same conditions but with fresh medium containing 100 mg of gentamicin per ml, monolayers were washed once with PBS (pH 7.4). During the kill period, all extracellular K. pneumoniae 03183 were killed but the viability of internalized bacteria was not affected. Finally, monolayers were washed twice with PBS and lysed with 0.1% Triton X-100 and the released intracellular bacteria were enumerated by plate count. The number of intracellular K. pneumoniae 03183 after invasion in the presence of an inhibitor was divided b...

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Section: Fig 1 Effects Of Different Inhibitors On the Internalizatimentioning

confidence: 66%

High Mobility Group Nucleosomal Binding Domain 2 Protein Protects Bladder Epithelial Cells from Klebsiella pneumoniae Invasion

Cao

Fan

et al. 2011

Biological & Pharmaceutical Bulletin

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“…Ratio measurements for phalloidin-to-DNase I ratios followed a modification of previously published methods (17). Phalloidin has been shown to be highly specific for F-actin, and DNase I was used to identify G-actin and discriminate it from F-actin (34). Images were acquired using a Kr/Ar laser (Bio-Rad Radiance 2000) scanning confocal microscope equipped with a Nikon X-60 (NA, 1.4) oil immersion objective.…”

Section: Methodsmentioning

confidence: 99%

“…The ratio of the value for phalloidin was divided by that for DNase I. The ratio values for the three ROIs for each cell were averaged, and the final number was taken as an index of the ratio of F-actin-to-G-actin (F/G ratio) for that cell as previously described (34). One advantage of the phalloidin/DNase I method used here is that the relative abundance of structures too small to resolve by confocal microscopy (such as G-actin monomers) can be quantitated by integrating the fluorescent signal.…”

Section: Methodsmentioning

confidence: 99%

“…To determine whether the above results could be reproduced with an independent second method with better time control, we also quick-fixed freshly enzymatically dissociated single muscle cells and costained them with fluorescent-labeled phalloidin to monitor the distribution of F-actin (red) and DNase I to monitor the distribution of G-actin (green) (34). DNase I, phalloidin, and merged images of illustrative individual stained cells are shown in Fig.…”

Section: Contractile Agonists Increase the F/g Ratio In Dvsmmentioning

confidence: 99%

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Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent

Kim¹,

Gallant²,

Leavis³

et al. 2008

American Journal of Physiology-Cell Physiology

116

165

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Dynamic remodeling of the actin cytoskeleton plays an essential role in the migration and proliferation of vascular smooth muscle cells. It has been suggested that actin remodeling may also play an important functional role in nonmigrating, nonproliferating differentiated vascular smooth muscle (dVSM). In the present study, we show that contractile agonists increase the net polymerization of actin in dVSM, as measured by the differential ultracentrifugation of vascular smooth muscle tissue and the costaining of single freshly dissociated cells with fluorescent probes specific for globular and filamentous actin. Furthermore, induced alterations of the actin polymerization state, as well as actin decoy peptides, inhibit contractility in a stimulus-dependent manner. Latrunculin pretreatment or actin decoy peptides significantly inhibit contractility induced by a phorbol ester or an α-agonist, but these procedures have no effect on contractions induced by KCl. Aorta dVSM expresses α-smooth muscle actin, β-actin, nonmuscle γ-actin, and smooth muscle γ-actin. The incorporation of isoform-specific cell-permeant synthetic actin decoy peptides, as well as isoform-specific probing of cell fractions and two-dimensional gels, demonstrates that actin remodeling during α-agonist contractions involves the remodeling of primarily γ-actin and, to a lesser extent, β-actin. Taken together, these results show that net isoform- and agonist-dependent increases in actin polymerization regulate vascular contractility.

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“…We compared the degree of polymerized actin for cells of young and old donors by dual fluorescence labeling using Alexa-647 phalloidin and Alexa-488 DNase I. Since the labeling of F-actin and G-actin with phalloidin and DNase I, respectively, is known to be specific and the staining patterns are thought to be spatially separate and distinct (43), this method allowed us to measure the relative amounts of G-and F-actin in the same cell simultaneously. As can be seen by confocal microscopy (Fig.…”

Section: Biophysical Journal 99(8) 2434-2442mentioning

confidence: 99%

Stiffening of Human Skin Fibroblasts with Age

Schulze

Wetzel

Kueper

et al. 2012

Clinics in Plastic Surgery

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Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods.

Cited by 84 publications

References 21 publications

High Mobility Group Nucleosomal Binding Domain 2 Protein Protects Bladder Epithelial Cells from Klebsiella pneumoniae Invasion

High Mobility Group Nucleosomal Binding Domain 2 Protein Protects Bladder Epithelial Cells from Klebsiella pneumoniae Invasion

Cytoskeletal remodeling in differentiated vascular smooth muscle is actin isoform dependent and stimulus dependent

Stiffening of Human Skin Fibroblasts with Age

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