Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells
Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells
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DOI：
10.1002/cm.10013
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发表时间：
2002-01-01
影响因子：
--
通讯作者：
Dawe, HR
中科院分区：
文献类型：
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作者：
Cramer, LP;Briggs, LJ;Dawe, HR
片状伪足突出与迁移成纤维细胞中肌动蛋白丝的分解有关 [Cramer, 1999: Curr.生物。 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、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- 和 F-肌动蛋白 [Mannherz et al., 1980: Eur. J.生物化学。 95:377-385]，当细胞在甲醛中固定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。在固定和透化过程中，92-98% 的肌动蛋白分子稳定保留在细胞中。此外，通过分别用latrunculin-A或jasplakinolide处理活细胞来增加或减少细胞G-肌动蛋白浓度，如预期的那样导致DNase I细胞染色强度相应增加和减少。 DNase I 荧光强度的这些变化准确地反映了在药物处理的活细胞制备的裂解物中独立测量的细胞 G-肌动蛋白浓度的增加和减少（回归系数 = 0.98）。这表明使用该方法进行 DNase I 细胞染色非常灵敏。应用这种方法，我们发现与非迁移成纤维细胞相比，迁移时片状基和紧邻片状基后面的宽带中 G-/F-肌动蛋白的比率较低。因此，我们预测迁移成纤维细胞中片状足的突出需要与丝分解紧密耦合，至少部分是因为 G-肌动蛋白在片状足内部和后面相对有限。这是第一份直接证明任何 G-肌动蛋白探针的细胞染色高灵敏度的报告，再加上荧光标记的 DNase I 的现成商业可及性，使其成为一种简单、方便且灵敏的 G-肌动蛋白细胞染色工具。 (C) 2002 Wiley-Liss, Inc.
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. (C) 2002 Wiley-Liss, Inc.