Comparative analysis of taxol-derived fluorescent probes to assess microtubule networks in a complex live three-dimensional tissue.
Comparative analysis of taxol-derived fluorescent probes to assess microtubule networks in a complex live three-dimensional tissue.
复制标题
对紫杉醇衍生的荧光探针进行比较分析，以评估复杂的活体三维组织中的微管网络。
DOI：
10.1002/cm.21599
复制
发表时间：
2020
期刊：
影响因子：
--
通讯作者：
McCartney,Brooke
中科院分区：
文献类型：
--
作者：
Logan,Gregory;McCartney,Brooke
果蝇卵子发生是一个很好的研究细胞骨架动力学的体内模型，因为在第10阶段结束时发生的快速细胞骨架重塑;然而，很少有强大的工具来检测活的复杂组织中的微管。最近开发的用于标记微管的膜渗透性紫杉醇荧光探针是重大技术进步，但这些探针的有效性和紫杉醇衍生物的潜在稳定作用尚未在体内得到很好的表征。在这里，我们比较了三种市售的紫杉醇衍生的微管标记物，以确定它们的功效和潜在的伪影。我们发现，所有三种探针标记微管的渗透性，亮度和信噪比的差异。然而，与紫杉醇一样，所有探针在较高浓度下都会破坏F-肌动蛋白细胞骨架。我们还发现，外排泵抑制剂维拉帕米增加了标记的强度，并适度增加了F‐actin缺陷的严重程度。在三种探针中，微管蛋白跟踪器（ThermoScientific）是最具渗透性的，并且是最亮的，具有最高的信噪比。此外，孵育30分钟后冲洗探针显著减少了F-肌动蛋白伪影，而不影响信号亮度。
Drosophila oogenesis is an excellent in vivo model for investigating cytoskeletal dynamics because of the rapid cytoskeletal remodeling that occurs at the end of stage 10; however, there are few robust tools for detecting microtubules in live complex tissues. The recent development of membrane permeable taxol‐based fluorescent probes to label microtubules is significant technical progress, but the effectiveness of these probes and the potential stabilizing effects of the taxol derivative have not been well characterized in vivo. Here, we compared three commercially available taxol‐derived microtubule labels to determine their efficacy and potential artifacts. We found that all three probes labeled microtubules with differences in permeability, brightness, and signal to noise ratio. Like taxol, however, all of the probes disrupted the F‐actin cytoskeleton at higher concentrations. We also found that the efflux pump inhibitor, verapamil, increased the intensity of the label and modestly increased the severity of the F‐actin defects. Of the three probes, Tubulin Tracker (ThermoScientific) was the most permeable and was brightest, with the highest signal to noise ratio. Furthermore, washing out the probe after a 30‐min incubation significantly reduced the F‐actin artifacts without compromising signal brightness.