A new method for direct detection of the sites of actin polymerization in intact cells and its application to differentiated vascular smooth muscle
A new method for direct detection of the sites of actin polymerization in intact cells and its application to differentiated vascular smooth muscle
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DOI：
10.1152/ajpcell.00210.2010
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发表时间：
2010-11-01
影响因子：
5.5
通讯作者：
Morgan, Kathleen G.
中科院分区：
文献类型：
--
作者：
Kim, Hak Rim;Leavis, Paul C.;Morgan, Kathleen G.
Kim HR, Leavis PC, Graceffa P, Gallant C, Morgan KG。一种直接检测完整细胞中肌动蛋白聚合位点的新方法及其在分化血管平滑肌中的应用。[J] .中国生物医学工程学报，2016,31(2):387 - 398。首次发表于2010年8月4日；doi: 10.1152 / ajpcell.00210.2010。在这里，我们报告并验证了一种新方法，广泛适用于分化细胞和组织，用于在生理条件下直接可视化肌动蛋白聚合。我们设计并测试了不同版本的荧光标记肌动蛋白，可逆地附着在蛋白质转导标签TAT上，并将这种新试剂引入完整分化的血管平滑肌细胞（dVSMCs）。将甘氨酸和半胱氨酸加入到TAT肽的nh2末端，形成一个硫代硝基苯甲酸酯加合物，即TAT- cys - s- stnb，合成了一种巯基反应型TAT肽。该肽很容易与g -肌动蛋白发生反应，并且该复合物迅速被新酶分离的dVSMC所吸收，正如TAT肽上FITC标记的荧光所示。通过比较不同版本的结构，我们确定了生物应用的最佳结构是与罗丹明标记的肌动蛋白偶联的非荧光标记TAT肽。当将tat - cys - s- stnb标记的罗丹明肌动蛋白（TSSAR）添加到活的、新鲜酶解的细胞中时，我们在细胞皮层观察到合并肌动蛋白的斑点。当罗丹明g -肌动蛋白加入到渗透细胞中时，这些点与我们之前报道的在相同细胞类型中出现的点无法区分。因此，这种新方法允许在不破坏天然状态的情况下将标记的g -肌动蛋白递送到完整细胞中，并将允许其进一步用于研究完整细胞中生理细胞内Ca2+浓度瞬态和信号转导对肌动蛋白动力学的影响。
Kim HR, Leavis PC, Graceffa P, Gallant C, Morgan KG. A new method for direct detection of the sites of actin polymerization in intact cells and its application to differentiated vascular smooth muscle. Am J Physiol Cell Physiol 299: C988-C993, 2010. First published August 4, 2010; doi: 10.1152/ajpcell.00210.2010.-Here we report and validate a new method, suitable broadly, for use in differentiated cells and tissues, for the direct visualization of actin polymerization under physiological conditions. We have designed and tested different versions of fluorescently labeled actin, reversibly attached to the protein transduction tag TAT, and have introduced this novel reagent into intact differentiated vascular smooth muscle cells (dVSMCs). A thiol-reactive version of the TAT peptide was synthesized by adding the amino acids glycine and cysteine to its NH2-terminus and forming a thionitrobenzoate adduct: viz. TAT-Cys-S-STNB. This peptide reacts readily with G-actin, and the complex is rapidly taken up by freshly enzymatically isolated dVSMC, as indicated by the fluorescence of a FITC tag on the TAT peptide. By comparing different versions of the construct, we determined that the optimal construct for biological applications is a nonfluorescently labeled TAT peptide conjugated to rhodamine-labeled actin. When TAT-Cys-S-STNB-tagged rhodamine actin (TSSAR) was added to live, freshly enzymatically isolated cells, we observed punctae of incorporated actin at the cortex of the cell. The punctae are indistinguishable from those we have previously reported to occur in the same cell type when rhodamine G-actin is added to permeabilized cells. Thus this new method allows the delivery of labeled G-actin into intact cells without disrupting the native state and will allow its further use to study the effect of physiological intracellular Ca2+ concentration transients and signal transduction on actin dynamics in intact cells.