活细胞成像，以评估在斜接主轴扰动后元相定时和细胞命运的动态
Summary
在这里，我们提出一个协议，以评估主轴形成和线粒体进展的动态。我们的延时成像应用使用户能够识别线粒体不同阶段的细胞，跟踪和识别线粒体缺陷，并在接触抗线粒药物时分析主轴动力学和线粒细胞命运。
Abstract
活细胞延时成像是细胞生物学中的重要工具，它提供了对细胞过程的洞察，否则这些细胞过程可能被固定细胞分析忽略、误解或误解。虽然固定细胞成像和分析是健壮的，足以观察细胞稳定状态，但它在定义细胞级事件的时间顺序时可能受到限制，并且无法评估动态过程的瞬态性质，包括线粒体进展。相反，活细胞成像是一种雄辩的工具，可用于观察细胞过程在单细胞水平随着时间的推移，并有能力捕捉过程的动态，否则在固定细胞成像中表现不佳。在这里，我们描述了一种生成带有荧光标记染色质和微管标记的细胞的方法，以及它们在活细胞成像方法中的使用，以监测元相染色体对齐和线粒出口。我们描述了基于成像的技术，以评估主轴形成和线粒进展的动态，包括识别线粒体不同阶段的细胞，识别和跟踪线粒缺陷，以及分析主轴动力学和使用线粒抑制剂治疗后的线粒细胞命运。
Introduction
基于图像的固定细胞分析通常用于评估细胞总体水平的变化，以响应各种扰动。当与细胞同步结合，然后采集和成像串行时间点时，这些方法可用于建议细胞事件序列。然而，固定细胞成像是有限的，因为时间关系是隐含在一个群体，而不是在单个细胞的水平证明。这样，虽然固定细胞成像和分析足以观察强健的表型和稳定状态变化，但检测随时间变化和仅影响细胞亚群的变化的能力并不完美。相反，活细胞成像是一个雄辩的工具，可用于观察单个细胞或细胞群中的细胞和亚细胞过程，随着时间的推移，无需辅助同步方法，这些方法本身可能会影响细胞行为1,2,3,4,5,6.
双相线轴的形成对于细胞分裂期间适当的染色体分离至关重要，从而产生两个基因相同的子细胞。线粒主轴结构的缺陷会破坏线粒体进展，并损害染色体分离的保真度，从而导致灾难性细胞分裂和细胞生存能力下降。因此，改变主轴形成的线粒体毒素是限制癌细胞7、8、9快速增殖的有希望的治疗方法。然而，在添加线粒毒物后，主轴结构的固定细胞分析在评估主轴形成的动态过程的能力方面受到限制，并且可能不能指示观察到的主轴结构变化是永久性的还是相反是暂时的，可以克服，以允许成功的细胞分裂。
在此协议中，我们描述了一种通过活细胞成像评估主轴扰动后线粒体动力学的方法。使用 hTERT 不朽的 RPE-1 细胞系，用于表达 RFP 标记的 Histone 2B 来可视化染色质，以及 EGFP 标记的 α-tubulin 来可视化微管、元相染色体对齐的时间、抗癫痫的发病，并最终线粒细胞的命运是使用染色体运动，压实和核形态的视觉线索评估。
Protocol
1. 生成 hTERT-RPE-1 细胞，可稳稳地表达 RFP-组蛋白 2B （RFP-H2B） 和 β-图布林-EGFP （tub-EGFP） 注：所有步骤均采用无菌技术，在生物安全 II+ （BSL2+） 安全柜中执行。 根据制造商对脂质转染输送系统的说明，通过转染293T细胞，使用适当的慢病毒质粒，生成携带相关基因（β-图布林-EGFP和RFP-H2B）的逆转录病毒。 第1天：使用一次性玻璃巴斯德移液器从一盘亚汇合293T细?…
Representative Results
在主轴扰动存在时线粒体进展的评估主轴聚焦的调节是正确双极主轴形成的关键步骤。通过蛋白质耗尽、药物抑制或中心体数改变破坏这一过程，破坏主轴结构，延迟或停止线粒体进展10、11、12、13。然而，一些扰动只是暂时延迟主轴的形成与细胞最终通过线?…
Discussion
延时成像提供的时间分辨率允许可视化和评估单个细胞内的顺序细胞事件。利用细胞同步，随后在连续时间点收集和固定细胞的方法是有限的，因为最终比较是在细胞群之间进行的。在细胞对扰动的反应可能不均匀，或者可视化过程是动态的环境下，活细胞延时成像更有能力跟踪和分析单个细胞的动态以及异质性在细胞群中。通过这种方式，延时成像在监测细胞通过线粒体分裂的动态阶段的进展?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
DLM 由 NSF GRFP 支持。ALM 由史密斯家族生物医学研究卓越奖资助。
Materials
| 0.05% Trypsin | Gibo-Life sciences | 25-510 | A serine protease used to release adherent cells from culture dishes |
| 15ml centrifuge tubes | Olympus Plastics | 28-101 | |
| 20x CFI Plan Fluor objective | Nikon | For use in Live-cell imaging to visualize both bright field and fluorescence | |
| 293T Cells | ATCC | CRL-3216 | For use in retroviral transfection; used in step 1.1 |
| a-tubulin-EGFP | Addgene | various numbers | Expression vector for alpha tubulin fused to a green fluorescent protein tag; for use in the visualization of tubulin in live-cell imaging: commercially available through addgene and other vendors |
| Alisertib | Selleckchem | S1133 | Small molecule inhibitor of the mitotic kinase Aurora A. Stock concentration is prepared at 10mM in DMSO, and used at a final concentration of 100nM. |
| Blasticidin | Invitrogen | A11139-03 | Antibiotic selection agent; used to select for a-tub-EGFP expressing cells |
| C02 | Airgas | For use in cell culture and live cell imaging | |
| Chroma ET-DS Red (TRITC/Cy3) | Chroma | 49005 | Single band filter set; excitation wavelength 545nm with 25nm bandwidth and emission at 605nm wavelength with 70nm bandwidth; for visualization of H2B-RFP |
| Chroma ET-EGFP (FITC/Cy2) | Chroma | 49002 | Single band filter set; excitation wavelength 470nm with 40nm bandwidth and emission at 525nm wavelength with 50nm bandwidth; for visualization of GFP-tubulin |
| disposable glass Pastuer pipets, sterilized | Fisher Scientific | 13-678-6A | For use in aspirating cells |
| Dulbecco’s Modified Eagle Medium (DMEM) | Gibo-Life sciences | 11965-084 | Cell culture medium for growth of RPE-1 and 293T cells |
| Fetal Bovine Serum (FBS) | Gibo-Life sciences | 10438-026 | Cell culture medium supplement |
| Lipofectamine 3000 and p3000 | Invitrogen | L3000-015 | Lipid based transfection reagent for transfection of plasmids; used in 1.1.4 |
| Multi well Tissue Culture dishes | Corning | various | for use in cell culture, transfection/infection, and live cell imaging |
| Nikon Ti-E microscope | Nikon | Inverted epifluorescence microscope for use in live-cell imaging | |
| NIS Elements HC | Nikon | Version 4.51 | Image acquisition and analysis software; used in sections 3 & 4 |
| OPTI-MEM | Gibo-Life sciences | 31985-070 | Reduced serum medium for cell transfection; used in step 1.1.3 |
| Penicillin/Streptomycin | Gibo-Life sciences | 15140-122 | antibiotic used in cell culture medium |
| phosphate bufferred saline (PBS) | Caisson labs | PBP06-10X1LT | sterile saline solution for use with cell culture |
| pMD2.G | Addgene | 12259 | Lentiviral VSV-G envelope expression construct; used in step 1.1.4 |
| Polybrene | Sigma-Aldrich | H9268 | Cationic polymer used to enhane viral infection efficiency; used in step 1.1.10 |
| psPAX | Addgene | 12260 | 2nd generation lentiviral packaging plasmid; used in step 1.1.4 |
| Puromycin | Invitrogen | ant-pr-1 | Antibiotic selection agent; used to select for RFP-H2B expressing cells |
| RFP- Histone 2B (H2B) | Addgene | various numbers | Expression vector for red fluorescent protein-tagged histone 2B; for use in the visualization of chromatin in live-cell imaging: commercially available through Addgene and other vendors |
| RNAi Max | Invitrogen | 13778-150 | Lipid based transfection reagent for transfection of siRNA constructs |
| RPE-1 cells | ATCC | CRL-4000 | Human retinal pigment epithelial cell line |
| Tissue culture dish 100x20mm | Corning | 353003 | for use in culturing adherent cells |
| Zyla sCMOS camera | Nikon | Camera attached to the micrscope, used for capturing images of cells |
References
- Szuts, D., Krude, T. Cell cycle arrest at the initiation step of human chromosomal DNA replication causes DNA damage. Journal of Cell Science. 117, 4897-4908 (2004).
- Gayek, A. S., Ohi, R. CDK-1 Inhibition in G2 Stabilizes Kinetochore-Microtubules in the following Mitosis. PLoS One. 11, e0157491 (2016).
- Mackay, D. R., Makise, M., Ullman, K. S. Defects in nuclear pore assembly lead to activation of an Aurora B-mediated abscission checkpoint. The Journal of Cell Biology. 191, 923-931 (2010).
- Cimini, D., Fioravanti, D., Salmon, E. D., Degrassi, F. Merotelic kinetochore orientation versus chromosome mono-orientation in the origin of lagging chromosomes in human primary cells. Journal of Cell Science. 115, 507-515 (2002).
- Kwon, M., et al. Mechanisms to suppress multipolar divisions in cancer cells with extra centrosomes. Genes & Development. 22, 2189-2203 (2008).
- Khodjakov, A., Rieder, C. L. Imaging the division process in living tissue culture cells. Methods. 38, 2-16 (2006).
- Gascoigne, K. E., Taylor, S. S. How do anti-mitotic drugs kill cancer cells?. Journal of Cell Science. 122, 2579-2585 (2009).
- van Vuuren, R. J., Visagie, M. H., Theron, A. E., Joubert, A. M. Antimitotic drugs in the treatment of cancer. Cancer Chemotherapy and Pharmacology. 76, 1101-1112 (2015).
- Chan, K. S., Koh, C. G., Li, H. Y. Mitosis-targeted anti-cancer therapies: where they stand. Cell Death and Diseases. 3, 411 (2012).
- Martin, M., Akhmanova, A. Coming into Focus: Mechanisms of Microtubule Minus-End Organization. Trends in Cell Biology. 28, 574-588 (2018).
- Maiato, H., Logarinho, E. Mitotic spindle multipolarity without centrosome amplification. Nature Cell Biology. 16, 386-394 (2014).
- Vitre, B. D., Cleveland, D. W. Centrosomes, chromosome instability (CIN) and aneuploidy. Current Opinion in Cell Biology. 24, 809-815 (2012).
- Godinho, S. A., Pellman, D. Causes and consequences of centrosome abnormalities in cancer. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 369, (2014).
- Navarro-Serer, B., Childers, E. P., Hermance, N. M., Mercadante, D., Manning, A. L. Aurora A inhibition limits centrosome clustering and promotes mitotic catastrophe in cells with supernumerary centrosomes. Oncotarget. 10, 1649-1659 (2019).
- Conte, N., et al. TACC1-chTOG-Aurora A protein complex in breast cancer. Oncogene. 22, 8102-8116 (2003).
- Schumacher, J. M., Ashcroft, N., Donovan, P. J., Golden, A. A highly conserved centrosomal kinase, AIR-1, is required for accurate cell cycle progression and segregation of developmental factors in Caenorhabditis elegans embryos. Development. 125, 4391-4402 (1998).
- Asteriti, I. A., Giubettini, M., Lavia, P., Guarguaglini, G. Aurora-A inactivation causes mitotic spindle pole fragmentation by unbalancing microtubule-generated forces. Molecular Cancer. 10, 131 (2011).
- Chan, J. Y. A clinical overview of centrosome amplification in human cancers. International Journal of Biological Sciences. 7, 1122-1144 (2011).
- Kramer, A., Maier, B., Bartek, J. Centrosome clustering and chromosomal (in)stability: a matter of life and death. Molecular Oncology. 5, 324-335 (2011).
- Ganem, N. J., Godinho, S. A., Pellman, D. A mechanism linking extra centrosomes to chromosomal instability. Nature. 460, 278-282 (2009).
- Silkworth, W. T., Nardi, I. K., Scholl, L. M., Cimini, D. Multipolar spindle pole coalescence is a major source of kinetochore mis-attachment and chromosome mis-segregation in cancer cells. PLoS One. 4, e6564 (2009).
- Nigg, E. A. Centrosome aberrations: cause or consequence of cancer progression?. Nature reviews, Cancer. 2, 815-825 (2002).
- Godinho, S. A., Kwon, M., Pellman, D. Centrosomes and cancer: how cancer cells divide with too many centrosomes. Cancer Metastasis Reviews. 28, 85-98 (2009).
- Quintyne, N. J., Reing, J. E., Hoffelder, D. R., Gollin, S. M., Saunders, W. S. Spindle multipolarity is prevented by centrosomal clustering. Science. 307, 127-129 (2005).
- Magidson, V., Khodjakov, A. Circumventing photodamage in live-cell microscopy. Methods in Cell Biology. 114, 545-560 (2013).
Cite This Article
Mercadante, D. L., Crowley, E. A., Manning, A. L. Live Cell Imaging to Assess the Dynamics of Metaphase Timing and Cell Fate Following Mitotic Spindle Perturbations. J. Vis. Exp. (151), e60255, doi:10.3791/60255 (2019).