Controlled ablation of microtubules using a picosecond laser
Controlled ablation of microtubules using a picosecond laser
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DOI：
10.1529/biophysj.104.049528
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发表时间：
2004-12-01
影响因子：
3.4
通讯作者：
Berns, MW
中科院分区：
文献类型：
--
作者：
Botvinick, EL;Venugopalan, V;Berns, MW
利用聚焦高强度光源进行烧蚀扰动已经成为细胞生物学和发育研究的重要技术。在靶向亚细胞结构中，许多研究必须处理不能确定靶向细胞器或大分子复合物的问题。在这里，我们证明了选择性地靶向微管为基础的结构与激光微束通过使用增强黄色荧光蛋白（EYFP）和增强青色荧光蛋白（ECFP）的变种的绿色荧光蛋白融合的小管的能力。产生稳定表达标记至微管蛋白α-亚基的EYFP和ECFP的Potorous tridactylus（PTK 2）细胞系。使用微管荧光作为指导，细胞用皮秒激光脉冲照射在细胞质和有丝分裂纺锤体中的离散微管位点。照射后1秒固定的细胞的相关薄切片透射电子显微照片表明，超微结构损伤的性质似乎是不同的EYFP和ECFP结构之间的不同，这表明不同的光子相互作用机制。我们的结论是，单细胞质和纺锤体微管的焦点中断可以精确控制相结合的激光微束照射与不同的荧光融合结构。详细讨论了可能的光子相互作用机制。
The use of focused high-intensity light sources for ablative perturbation has been an important technique for cell biological and developmental studies. In targeting subcellular structures many studies have to deal with the inability to target, with certainty, an organelle or large macromolecular complex. Here we demonstrate the ability to selectively target microtubule-based structures with a laser microbeam through the use of enhanced yellow fluorescent protein (EYFP) and enhanced cyan fluorescent protein (ECFP) variants of green fluorescent protein fusions of tubule. Potorous tridactylus (PTK2) cell lines were generated that stably express EYFP and ECFP tagged to the alpha-subunit of tubulin. Using microtubule fluorescence as a guide, cells were irradiated with picosecond laser pulses at discrete microtubule sites in the cytoplasm and the mitotic spindle. Correlative thin-section transmission electron micrographs of cells fixed one second after irradiation demonstrated that the nature of the ultrastructural damage appeared to be different between the EYFP and the ECFP constructs suggesting different photon interaction mechanisms. We conclude that focal disruption of single cytoplasmic and spindle microtubules can be precisely controlled by combining laser microbeam irradiation with different fluorescent fusion constructs. The possible photon interaction mechanisms are discussed in detail.