A simple fixation procedure for immunofluorescent detection of different cytoskeletal components within the same cell

Vielkind, Ursula; Swierenga, S.H.H.

doi:10.1007/bf00501916

Cited by 88 publications

(43 citation statements)

References 28 publications

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“…Immunofluorescence Staining-Immunofluorescence was performed according as described (30) with minor changes (14). Cells were fixed using 3.7% formaldehyde, and the rehydrated samples were incubated overnight at 4°C with primary antibody anti-HuR diluted 1:300 in blocking buffer and then incubated with secondary antibody conjugated with Alexa Fluor-594 (Molecular Probes, Eugene, OR) for 2 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Polyamine Depletion Increases Cytoplasmic Levels of RNA-binding Protein HuR Leading to Stabilization of Nucleophosmin and p53 mRNAs

Zou¹,

Mazan-Mamczarz

Rao³

et al. 2006

Journal of Biological Chemistry

116

167

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Polyamines are essential for maintaining normal intestinal epithelial integrity, an effect that relies, at least in part, on their ability to keep low levels of nucleophosmin (NPM) and p53 mRNAs. The RNA-binding protein HuR associates with the p53 mRNA, as reported previously, and with the NPM mRNA, computationally predicted to be a target of HuR. Here, we show that HuR binds the NPM and p53 3-untranslated regions and stabilizes these mRNAs in polyaminedepleted intestinal epithelial cells. Depletion of cellular polyamines by inhibiting ornithine decarboxylase with ␣-difluoromethylornithine dramatically enhanced the cytoplasmic abundance of HuR, whereas ectopic ornithine decarboxylase overexpression decreased cytoplasmic HuR; neither intervention changed whole-cell HuR levels. HuR was found to specifically bind the 3-untranslated regions of NPN and p53 mRNAs. HuR silencing rendered the NPM and p53 mRNAs unstable and prevented increases in NPM and p53 mRNA and protein in polyamine-deficient cells. These results indicate that polyamines modulate cytoplasmic HuR levels in intestinal epithelial cells, in turn controlling the stability of the NPM and p53 mRNAs and influencing NPM and p53 protein levels.

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Section: Methodsmentioning

confidence: 99%

Polyamine Depletion Increases Cytoplasmic Levels of RNA-binding Protein HuR Leading to Stabilization of Nucleophosmin and p53 mRNAs

Zou¹,

Mazan-Mamczarz

Rao³

et al. 2006

Journal of Biological Chemistry

116

167

View full text Add to dashboard Cite

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“…The coverslips were then transferred to DMEMþHEPES prewarmed to 37 C. After 5, 10, or 15 min, the coverslips were plunged into ice-cold Hanks' balanced salt solution for 1 min. The cells were fixed in formaldehyde PEM buffer (100 mM PIPES, 5 mM EGTA, 2 mM MgCl 2 , 4% v/v methanol-free formaldehyde, 0.2% v/v Triton X-100) for 10 min at room temperature (31). The slides were gently washed with PBS, soaked in 1 mg/mL BSA in PBS (BSA buffer) for 5 min, and then stained with 0.6 mM phalloidin-atto488 (49409, Sigma) in BSA buffer for 40 min at 4 C. Finally, the samples were rinsed with PBS and mounted in ProLong Diamond Antifade with DAPI (P36962, ThermoFisher Scientific, Waltham, MA).…”

Section: Structured Illumination Microscopymentioning

confidence: 99%

“…These intervals were chosen because they roughly correspond to the rapid-spreading, peak contact area, and contraction periods, respectively. To ensure that cytoskeletal structure was not altered by the fixation and staining process, those steps were carried out in PEM buffer, which has been shown to preserve F-actin and microtubule structure (31).…”

Section: F-actin Reorganizes During Fpmentioning

confidence: 99%

Frustrated Phagocytic Spreading of J774A-1 Macrophages Ends in Myosin II-Dependent Contraction

Kovari

Wei

Chang

et al. 2016

Biophysical Journal

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Conventional studies of dynamic phagocytic behavior have been limited in terms of spatial and temporal resolution due to the inherent three-dimensionality and small features of phagocytosis. To overcome these issues, we use a series of frustrated phagocytosis assays to quantitatively characterize phagocytic spreading dynamics. Our investigation reveals that frustrated phagocytic spreading occurs in phases and is punctuated by a distinct period of contraction. The spreading duration and peak contact areas are independent of the surface opsonin density, although the opsonin density does affect the likelihood that a cell will spread. This reinforces the idea that phagocytosis dynamics are primarily dictated by cytoskeletal activity. Structured illumination microscopy reveals that F-actin is reorganized during the course of frustrated phagocytosis. F-actin in early stages is consistent with that observed in lamellipodial protrusions. During the contraction phase, it is bundled into fibers that surround the cell and is reminiscent of a contractile belt. Using traction force microscopy, we show that cells exert significant strain on the underlying substrate during the contraction phase but little strain during the spreading phase, demonstrating that phagocytes actively constrict during late-stage phagocytosis. We also find that latestage contraction initiates after the cell surface area increases by 225%, which is consistent with the point at which cortical tension begins to rise. Moreover, reducing tension by exposing cells to hypertonic buffer shifts the onset of contraction to occur in larger contact areas. Together, these findings provide further evidence that tension plays a significant role in signaling late-stage phagocytic activity.

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“…purpuratus embryos were fixed for 5 minutes in PEM buffer (Vielkind and Swierenga, 1989) or ice-cold methanol. Embryos were washed with PBS and probed with primary antibody diluted in SuperBlock (Thermo).…”

Section: Immunofluorescence Microscopymentioning

confidence: 99%

Eph-Ephrin signaling and focal adhesion kinase regulate actomyosin-dependent apical constriction of ciliary band cells

Krupke

Burke

2014

Journal of Cell Science

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Apical constriction typically accompanies inward folding of an epithelial sheet. In recent years there has been progress in understanding mechanisms of apical constriction and their contribution to morphogenetic processes. Sea urchin embryos form a specialized region of ectoderm, the ciliary band, which is a strip of epithelium, three to five cells wide, encircling the oral ectoderm and functioning in larval swimming and feeding. Ciliary band cells exhibit distinctive apical-basal elongation, have narrow apices bearing a cilium, and are planar polarized, so that cilia beat away from the mouth. Here, we show that filamentous actin and phosphorylated myosin light chain are uniquely distributed in ciliary band cells. Inhibition of myosin phosphorylation or actin polymerization perturbs this distribution and blocks apical constriction. During ciliary band formation, Sp-Ephrin and Sp-Eph expression overlap in the presumptive ciliary band. Knockdown of Sp-Eph or Sp-Ephrin, or treatment with an Eph kinase inhibitor interferes with actomyosin networks, accumulation of phosphorylated FAK (pY 397 FAK), and apical constriction. The cytoplasmic domain of Sp-Eph, fused to GST and containing a single amino acid substitution reported as kinase dead, will pull down pY 397 FAK from embryo lysates. As well, pY 397 FAK colocalizes with Sp-Eph in a JNK-dependent, planar polarized manner on latitudinal apical junctions of the ciliary band and this polarization is dissociable from apical constriction. We propose that Sp-Eph and pY 397 FAK function together in an apical complex that is necessary for remodeling actomyosin to produce centripetal forces causing apical constriction. Morphogenesis of ciliary band cells is a unique example of apical constriction in which receptor-mediated cell shape change produces a strip of specialized tissue without an accompanying folding of epithelium.

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A simple fixation procedure for immunofluorescent detection of different cytoskeletal components within the same cell

Cited by 88 publications

References 28 publications

Polyamine Depletion Increases Cytoplasmic Levels of RNA-binding Protein HuR Leading to Stabilization of Nucleophosmin and p53 mRNAs

Polyamine Depletion Increases Cytoplasmic Levels of RNA-binding Protein HuR Leading to Stabilization of Nucleophosmin and p53 mRNAs

Frustrated Phagocytic Spreading of J774A-1 Macrophages Ends in Myosin II-Dependent Contraction

Eph-Ephrin signaling and focal adhesion kinase regulate actomyosin-dependent apical constriction of ciliary band cells

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