In vivo selective cytoskeleton dynamics quantification in interphase cells induced by pulsed ultraviolet laser nanosurgery
In vivo selective cytoskeleton dynamics quantification in interphase cells induced by pulsed ultraviolet laser nanosurgery
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DOI：
10.1111/j.1600-0854.2005.00334.x
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发表时间：
2005-12-01
期刊：
影响因子：
4.5
通讯作者：
Stelzer, EHK
中科院分区：
文献类型：
--
作者：
Colombelli, J;Reynaud, EG;Stelzer, EHK
我们报告的操纵细胞内细丝使用纳米手术系统的基础上优化的局部切断生物聚合物的亚纳秒脉冲紫外激光。通过诱导选定的微管（MT）的人工灾难，我们在整个细胞的间期Ptk-2细胞进行收缩率测量。我们量化了两种标记方法和三种荧光标记物的影响，与罗丹明和黄色荧光蛋白（YFP）微管蛋白相比，Alexa-488微管蛋白的解聚速度快25%，与稳定转染相比，显微注射诱导的变异性高20%。使用EB 3-GFP作为尖端标记物，我们建立了一种新的协议，以测量收缩率，生长率和拯救频率同时在活细胞中具有高的时间和空间特异性。正如我们的分析表明，激光诱导MT动力学是生理相关的。该方法在测量事件数量方面提供的高统计效率以及因此降低的标准偏差代表了体内动态不稳定参数测量的重要定量改进。我们扩展的应用程序的方法，通过演示诱导的动态行为的肌动蛋白应力纤维切断后。这种新的方法使定量调查的细胞骨架动力学的局部限制。
We report on the manipulation of intracellular filaments using a nanosurgery system based on a subnanosecond pulsed UV laser optimized for the localized severing of biological polymers. By inducing artificial catastrophe of selected microtubules (MTs), we perform shrinkage-rate measurements in interphase Ptk-2 cells throughout the entire cell. We quantify the impact of two labeling methods and three fluorescent markers, showing a 25% faster depolymerization with Alexa-488 tubulin compared with Rhodamine and yellow fluorescent protein (YFP) tubulins and a 20% higher variability induced by microinjection compared with stable transfection. Using EB3-GFP as a tip marker, we establish a new protocol to measure shrinkage rate, growth rate and rescue frequency simultaneously with high temporal and spatial specificity in live cells. As our analysis shows, laser-induced MT dynamics are physiologically relevant. The high statistical efficiency that the method offers in terms of numbers of measured events and therefore reduced standard deviations represents an important quantitative improvement in the measurement of dynamic instability parameters in vivo. We extend the application of the method by demonstrating induced dynamic behavior of actin-stress fibers after severing. This new method enables the quantitative investigation of cytoskeleton dynamics in a local confinement.