European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Meyerhofstrasse 1, Heidelberg, Germany.
Cytoskeleton (Hoboken). 2011 May;68(5):266-78. doi: 10.1002/cm.20510. Epub 2011 Apr 13.
The genetic integrity of every organism depends on the faithful partitioning of its genome between two daughter cells in mitosis. In all eukaryotes, chromosome segregation requires the assembly of the mitotic spindle, a bipolar array of dynamic microtubules. Perturbations in microtubule dynamics affect spindle assembly and maintenance and ultimately result in aberrant cell divisions. To identify new regulators of microtubule dynamics within the hundreds of mitotic hits, reported in RNAi screens performed in C. elegans, Drosophila and mammalian tissue culture cells [Sonnichsen et al., 2005; Goshima et al., 2007; Neumann et al., 2010], we established a fast and quantitative assay to measure microtubule dynamics in living cells. Here we present a fully automated workflow from RNAi transfection, via image acquisition and data processing, to the quantitative characterization of microtubule behaviour. Candidate genes are knocked down by solid-phase reverse transfection with siRNA oligos in HeLa cells stably expressing EB3-EGFP, a microtubule plus end marker. Mitotic cells are selected using an automatic classifier [Conrad et al., 2011] and imaged on a spinning disk confocal microscope at high temporal and spatial resolution. The time-lapse movies are analysed using a multiple particle tracking software, developed in-house, that automatically detects microtubule plus ends, tracks microtubule growth events over consecutive frames and calculates growth speeds, lengths and lifetimes of the tracked microtubules. The entire assay provides a powerful tool to analyse the effect of essential mitotic genes on microtubule dynamics in living cells and to dissect their contribution in spindle assembly and maintenance.
每个生物体的遗传完整性都依赖于其基因组在有丝分裂过程中在两个子细胞之间的忠实分配。在所有真核生物中，染色体分离需要有丝分裂纺锤体的组装，这是一个动态微管的双极阵列。微管动力学的扰动会影响纺锤体的组装和维持，最终导致异常的细胞分裂。为了在 RNAi 筛选中鉴定出数百个有丝分裂命中的新的微管动力学调节剂，在秀丽隐杆线虫、果蝇和哺乳动物组织培养细胞中进行了报道[ Sonnichsen 等人，2005 年；Goshima 等人，2007 年；Neumann 等人，2010 年]，我们建立了一种快速和定量的测定方法，用于测量活细胞中的微管动力学。在这里，我们提出了一个从 RNAi 转染到图像采集和数据处理的全自动工作流程，以对微管行为进行定量表征。候选基因通过固相反义寡核苷酸转染在稳定表达 EB3-EGFP 的 HeLa 细胞中被敲低，EB3-EGFP 是微管的正极标记物。有丝分裂细胞使用自动分类器[ Conrad 等人，2011 年]进行选择，并在高速时空分辨率的旋转盘共聚焦显微镜上成像。使用我们内部开发的多粒子跟踪软件对延时电影进行分析，该软件自动检测微管正极，在连续的帧上跟踪微管生长事件，并计算跟踪微管的生长速度、长度和寿命。整个测定为分析必需的有丝分裂基因对活细胞中微管动力学的影响以及剖析它们在纺锤体组装和维持中的贡献提供了一个强大的工具。
