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首页> 外文期刊>Cell motility and the cytoskeleton >Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells.
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Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells.

机译：使用荧光标记的脱氧核糖核酸酶I在空间上测量迁移和非迁移细胞中的G-肌动蛋白水平。

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Lamellipodium protrusion is linked to actin filament disassembly in migrating fibroblasts [Cramer, 1999: Curr. Biol. 9:1095-1105]. To further study this relationship, we have identified a method to specifically and sensitively detect G-actin in distinct spatial locations in motile cells using deoxyribonuclease I (DNase I). Although DNase I can bind both G- and F-actin in vitro [Mannherz et al., 1980: Eur. J. Biochem. 95:377-385], when cells were fixed in formaldehyde and permeabilized in detergent, fluorescently-labelled DNase I specifically stained G-actin and not F-actin. 92-98% of actin molecules were stably retained in cells during fixation and permeabilization. Further, increasing or decreasing cellular G-actin concentration by treating live cells with latrunculin-A or jasplakinolide, respectively, caused a respective increase and decrease in DNase I cell-staining intensity as expected. These changes in DNase I fluorescence intensity accurately reflected increases and decreases in cellular G-actin concentration independently measured in lysates prepared from drug-treated live cells (regression coefficient = 0.98). This shows that DNase I cell-staining is very sensitive using this method. Applying this method, we found that the ratio of G-/F-actin is lower in both the lamellipodium and in a broad band immediately behind the lamellipodium in migrating compared to non-migrating fibroblasts. Thus, we predict that protrusion of the lamellipodium in migrating fibroblasts requires tight coupling to filament disassembly at least in part because G-actin is relatively limited within and behind the lamellipodium. This is the first report to directly demonstrate high sensitivity of cell-staining for any G-actin probe and this, together with the ready commercial accessibility of fluorescently-labelled DNase I, make it a simple, convenient, and sensitive tool for cell-staining of G-actin. Copyright 2002 Wiley-Liss, Inc.
机译：层状脂质体的突出与迁移的成纤维细胞中肌动蛋白丝的分解有关[Cr​​amer，1999：Curr。生物学9：1095-1105]。为了进一步研究这种关系，我们确定了一种使用脱氧核糖核酸酶I（DNase I）特异性和灵敏地检测运动细胞中不同空间位置的G-肌动蛋白的方法。尽管DNase I可以在体外结合G-肌动蛋白和F-肌动蛋白[Mannherz et al。，1980：Eur。 J.生物化学。 95：377-385]，当细胞固定在甲醛中并在去污剂中透化时，荧光标记的DNase I特异性染色G-肌动蛋白而不是F-肌动蛋白。 92-98％的肌动蛋白分子在固定和通透过程中稳定保留在细胞中。此外，通过分别用latrunculin-A或jasplakinolide处理活细胞来增加或降低细胞中G-肌动蛋白的浓度，会分别导致DNase I细胞染色强度的增加和减少。 DNase I荧光强度的这些变化准确反映了在用药物处理的活细胞制备的裂解物中独立测量的细胞中G-肌动蛋白浓度的增加和减少（回归系数= 0.98）。这表明使用这种方法对DNase I细胞染色非常敏感。应用这种方法，我们发现与非迁移的成纤维细胞相比，在迁移中的lamellipodium和紧接在lamellipodium后面的宽带中，G- / F-肌动蛋白的比率都较低。因此，我们预测，在迁移的成纤维细胞中，lamellipodium的突出需要紧密耦合到细丝分解，至少部分是因为在lamellipodium内部和后面相对有限的G-肌动蛋白。这是第一个直接证明细胞染色对任何G-肌动蛋白探针都具有高灵敏度的报告，这与荧光标记的DNase I的现成商业可得性一起，使其成为用于细胞染色的简单，方便且敏感的工具肌动蛋白。版权所有2002 Wiley-Liss，Inc.

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