This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.
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.
在细胞分裂的基因组中的平等隔离需要复制的DNA和纺锤体微管之间的正确交互。与染色体通过蛋白质的合奏组装在当时被称为动粒1丝点微管的物理交互。染色体正确分配要求姐姐动粒是双取向,其中每个姊妹与微管从相对纺锤体极始发相关联。动粒的微管(克拉-MT)附件的未在双取向构象被快速,有效地去稳定提供的机会,以建立biorientation在称为误差校正的处理。以前在哺乳动物细胞中建立了纠错法5要求使用可逆的小分子抑制剂对Eg5的(驱动蛋白5)装配单极纺锤体。药物治疗产生了许多错误syntelic附件,其中机器人^ h姐妹动粒附着于同一主轴极点。药物的后续冲洗允许误差校正处理的可视化。纠错测定可以做的小分子抑制剂或击倒的存在下,研究以校正错误克拉-MT附件候选蛋白的贡献。
可视化在活细胞中的纠错的能力是一个强大的工具,以进一步了解参与这一复杂过程的分子机制。然而,大量存在于大多数细胞系的染色体形成了挑战中观察个体克拉-MT附件。因为它们含有少至4个染色体6,但小分子抑制剂的果蝇S2细胞将是理想的用于施加纠错测定驱动蛋白5如S-三苯甲基-L-半胱氨酸(STLC)和monastrol 7-9不影响在果蝇细胞主轴组件或驱动蛋白5的运动功能。因此,我们属泰德下诱导型启动子也就是驱动蛋白5抑制剂敏感表达人驱动蛋白-5的果蝇S2细胞系。这个协议描述如何拦截内源性果蝇驱动蛋白5同系物,Klp61F,并使用该细胞系中的细胞系的纠错检测。
Visualizing error correction is a valuable technique to study the steps involved in this important and complex cellular process. To do so, erroneous attachments are generated using reversible inhibitors, and error correction is observed upon washout of the drug. This assay was originally developed using mammalian tissue culture cells5. However the presence of large number of kinetochores in many model mammalian cell types poses a challenge in observing individual error correction events. Drosophila S2 cells possess as few as 4 pairs of kinetochores, making them a more preferable cell line for observing error correction. However, a major drawback is that many inhibitors are ineffective in Drosophila S2 cells. Thus, the ability to generate a humanized Drosophila S2 cell line expressing human kinesin-5 provides a valuable tool to study error correction.
Although Drosophila S2 cells can be a better cell line to study error correction, the multiple steps involved in obtaining the cell line and knocking down essential genes poses some challenges to this technique. For instance, transfection efficiency can be quite low. If less than 20% of the cells are expressing the Eg5-mCherry, the transfection should be repeated as the selection process will take longer. Also, the percentage of cells expressing both fluorescent proteins may decrease over time. This can be overcome by splitting cells in the presence of Blasticidin S HCl and Hygromycin B to select for cells expressing Eg5-mCherry and GFP-α-tubulin, respectively. It is also important to note that Drosophila S2 cells have orthologues to many human proteins and; therefore, it is critical to optimize the knockdown conditions for the endogenous proteins. Optimal knockdown conditions may vary in the amount of dsRNA and the length of treatment. Considering the emergence and rapid improvement of CRISPR-Cas9 technologies15-17, generation of a Drosophila cell line with the Klp61F gene replaced by human Eg5 presents a powerful alternative that would overcome the limitations of the transfection and knockdown approach. Our work demonstrates that fly-to-human gene replacement should be a viable option in this case although the necessary reagents to do so in Drosophila S2 cells are currently being developed.
This procedure is not limited to Eg5, but can be applied to study the function of other proteins of interest. If using inhibitors that have previously been established to be ineffective in Drosophila S2 cells, this protocol can be modified to study the direct effect of inhibitors in live cells without concerns about off target effects. The cell line produced using this protocol could also be used in high-throughput screening analyses to identify potential drugs targeting proteins involved in error correction.
The authors have nothing to disclose.
We would like to thank Patricia Wadsworth for the gift of the kinesin-5 construct. This work was supported by an NIH grant (5 R01 GM107026) to T.J.M. and by Research Grant No. 5-FY13-205 from the March of Dimes Foundation to T.J.M., as well as support from the Charles H. Hood Foundation, Inc., Boston, MA. to T.J.M.
Effectine Transfection Reagent | Qiagen | 301425 | |
Klp61F cDNA | Drosophila Genomic Resource Center | 13690 | Gene Name LD15641 |
Schneider’s Media | Invitrogen | 21720-024 | |
Fetal Bovine Serum, certified | Invitrogen | 10082-147 | Heat Inactivated, US origin |
Copper (II) Sulfate (CuSO4) | Sigma | C8027-500G | 500mM stock |
S-trityl-l-cysteine | Sigma | 164739-5G | 1mM stock in DMSO |
Blasticidin HCl | Invitrogen | R21001 | 5mg/ml stock in 1x PBS |
Hygromycin B | Invitrogen | 10687010 | |
T7 Large scale RNA Production System | Promega | P1320 | The approximate dsRNA concentration from each reaction is about 5-15µg/µl |
Klp61F RNAi F primer | Invitrogen | TAATACGACTCACTATAGGGTA-TTTGCGCATTATTTTAAAA | |
Klp61F RNAi R primer | Invitrogen | TAATACGACTCACTATAGGGAT-ATTGATCAATTGAAAC | |
PCR clean-up kit | Mo Bio Laboratories, Inc | 1250 | |
Concanavalin A | Sigma | C5275 | 0.5mg/ml solution made by dissolving in 1xPBS. |
Boiled Donkey Serum | Jackson ImmunoResearch Labs | 017-000-121 | 5% stock solution in 1X PHEM buffer, bring solution up to boil. Stored in 4°C. |
Mounting Media | 20mM Tris pH 8.0, 0.5% N-propyl gallate, 90% Glycerol. Stored in 4°C | ||
1x BRB-80 | 80 mM PIPES pH 6.9; 1 mM EGTA; 1 mM MgCl2 | ||
White Light Source | Lumencor Inc | ||
ET EGFP Filter Cube | Chroma | 49002 | |
DSRed Filter Cube | Chroma | 49005 | |
anti-NDC80 antibody | Custom made by the Maresca Lab | ||
DM1α (anti-α-tubulin) | Sigma | T6199 | |
anti-CID antibody | AbCam | ab10887 |