Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5
Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5
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DOI：
10.3791/53594
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发表时间：
2016-01-01
影响因子：
1.2
通讯作者：
Maresca, Thomas J.
中科院分区：
文献类型：
--
作者：
Ye, Anna A.;Maresca, Thomas J.
着丝点是在细胞分裂过程中聚集在着丝粒上并将染色体连接到纺锤体微管的大型蛋白质结构。遗传物质的正常分布要求每条染色体上的姐妹着丝点在进入后期之前，通过附着在相对纺锤体极点的微管上而变成双取向。然而，错误的、非生物定向的附着状态是常见的，并且在细胞分裂过程中存在检测和纠正这种附着的细胞途径。不适当的着丝蛋白-微管相互作用被破坏的过程称为纠错。为了研究活细胞中的误差校正，通过化学抑制kinesin-5马达，故意产生不正确的附着物，导致单极主轴组装，并在药物冲洗后观察到从错误取向到双向取向的转变。在许多模型组织培养细胞类型中，大量的染色体对观察个体错误纠正事件提出了挑战。果蝇S2细胞是更好的研究对象，因为它们只有4对染色体。然而，小分子激酶5抑制剂对果蝇激酶5 （Klp61F）无效。在这里，我们描述了如何建立一个果蝇细胞系，有效地取代Klp61F与人类运动蛋白-5，这使得细胞对运动的药理抑制敏感，适合用于基于细胞的误差校正实验。
Kinetochores are large protein-based structures that assemble on centromeres during cell division and link chromosomes to spindle microtubules. Proper distribution of the genetic material requires that sister kinetochores on every chromosome become bioriented by attaching to microtubules from opposite spindle poles before progressing into anaphase. However, erroneous, non-bioriented attachment states are common and cellular pathways exist to both detect and correct such attachments during cell division. The process by which improper kinetochore-microtubule interactions are destabilized is referred to as error correction. To study error correction in living cells, incorrect attachments are purposely generated via chemical inhibition of kinesin-5 motor, which leads to monopolar spindle assembly, and the transition from mal-orientation to biorientation is observed following drug washout. The large number of chromosomes in many model tissue culture cell types poses a challenge in observing individual error correction events. Drosophila S2 cells are better subjects for such studies as they possess as few as 4 pairs of chromosomes. However, small molecule kinesin-5 inhibitors are ineffective against Drosophila kinesin-5 (Klp61F). Here we describe how to build a Drosophila cell line that effectively replaces Klp61F with human kinesin-5, which renders the cells sensitive to pharmacological inhibition of the motor and suitable for use in the cell-based error correction assay.