无细胞爪卵萃取物微乳液细胞周期振荡的重建
Summary
本文提出了在油中微乳液中对爪蟾卵提取物进行单细胞水平的体外自持续有丝分裂振荡的生成方法。
Abstract
实时测量单细胞水平的振荡, 对于揭示生物钟的机理具有重要意义。虽然从爪蟾卵制备的大块提取物在细胞周期发展的基础上解剖生物化学网络中具有强大的功能, 但它们的整体平均测量通常会导致阻尼振荡, 尽管每个单个振荡器被持续。这是由于噪声生物系统中单个振荡器之间的完美同步的困难。为了检索振荡器的单细胞动力学, 我们开发了一个以液滴为基础的人工细胞系统, 它能在细胞状腔内重组爪蟾卵的循环细胞质提取物的有丝分裂周期。这些简单的胞浆细胞表现出30多个周期的持续振荡。为了构建更复杂的细胞核细胞, 我们添加了 demembranated 精子染色质, 以触发系统中的核自组装。我们观察到染色体凝结/decondensation 和细胞核包络破裂/改革的周期性进展, 就像在真实细胞中。这表明有丝分裂振荡器的功能忠实地推动多个下游有丝分裂事件。利用多通道时程荧光显微镜, 我们同时跟踪了单个液滴的有丝分裂振荡器和下游过程的动力学。人工细胞周期系统提供了一个高通量的框架, 用于定量操作和分析单细胞分辨率的有丝分裂振荡, 这可能提供了重要的洞察力的调节机制和功能时钟。
Introduction
由爪蟾卵制备的细胞质提取物是细胞周期生化研究中最主要的模型之一, 考虑到卵母细胞的体积大、细胞周期的快速发展以及重组的能力。体外有丝分裂事件1,2。该系统允许初步发现和机械特性的关键细胞周期调节器, 如成熟促进因子 (MPF) 以及下游有丝分裂过程, 包括主轴组装和染色体分离1 ,2,3,4,5,6,7,8,9,10, 11。爪蟾卵提取物也被用来详细解剖细胞周期时钟8,12,13,14的调节网络和 DNA 损伤的研究/复制检查点15和有丝分裂主轴组件检查点 16、17、18。
这些研究的细胞周期使用爪蟾卵提取物主要是基于散装测量。然而, 常规的散装反应化验可能不会模仿真实的细胞行为, 因为它们的维度和亚细胞空间划分的反应分子的主要差异。此外, 在快速阻尼8之前, 对有丝分裂活动的大量测量往往会给出有限的周期数。大量反应的缺点使萃取系统无法进一步了解复杂时钟的动态特性和功能。最近的研究已经封装了无细胞 cytostatic 因子 (CSF)爪蟾提取物 19,20到大小定义的细胞样车厢, 这有助于阐明如何调整主轴尺寸细胞质容积。然而, 这种体外系统是在减数分裂的中期被逮捕的 cytostatic 因子1的作用, 一个能够长期持续振荡的单细胞水平, 以进一步调查细胞周期振荡 器。
为了研究细胞周期振荡的单细胞分辨率, 我们开发了一个细胞规模, 高通量系统的重建和同时测量多个自持续有丝分裂振荡过程中的个别微乳液液 滴。在详细的视频协议中, 我们演示了在10到300µm 的微乳液中封装循环爪蟾卵胞浆的人工有丝分裂振荡系统的建立。在该系统中, 有丝分裂振荡包括染色体凝结和脱凝、核包络击穿和改造, 以及后期基质的降解和合成 (如本协议中的 securin mCherry) 是成功重组。
Protocol
这里描述的所有方法都得到了密歇根大学机构动物护理和使用委员会 (IACUC) 的批准。 1. 细胞周期重建和检测材料的制备 securin-mCherry 质粒 DNA 构建及 mRNA 纯化的吉布森组装克隆 通过聚合酶链反应 (PCR) 和凝胶纯化21,22, 制备三个 DNA 片段, 包括 pMTB2 向量主干、securin 和 mCherry。 使用 fluorospectrometer 测?…
Representative Results
在图 2中, 我们表明该协议在简单的无核细胞和复杂的细胞核中产生有丝分裂振荡, 振荡器驱动原子核形成和变形的循环交替。无核的水滴产生的有丝分裂振荡高达30无阻尼吸循环超过92小时的时间跨度, 这表明由周期性合成和退化的荧光记者 securin-mCherry (图 2A)。Securin 是后期促进复合 APC/C 的基底。securin-mCherry 在后期的降解从而?…
Discussion
我们提出了一种新的方法来开发一个高通量人工细胞系统, 使体外重建和长期跟踪自我持续的细胞周期振荡的单细胞水平。有几个关键步骤使此方法成功。首先, 与下蛋相比, 新鲜挤压的爪蛋具有良好的品质, 往往会产生具有更持久振荡活性的萃取物。其次, 在表面活性剂稳定的微环境中, 萃取物的封装对于保持振荡活性至关重要。第三, 将油滴孵化成一根薄薄的管子, 将水滴限制在一层…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们感谢玛德琳路建设 securin-mCherry 质粒, 圈人李, 肯尼斯和艾伦 P 刘讨论关于水滴生成, 杰里米 b. 张和詹姆斯 e. 费雷尔 Jr 提供 GFP-NLS 结构。这项工作得到了国家科学基金会 (早期职业补助金 #1553031)、国立卫生研究院 (米拉 #GM119688) 和斯隆研究金的支持。
Materials
| Xenopus laevis frogs | Xenopus-I Inc. | ||
| QIAprep spin miniprep kit | QIAGEN | 27104 | |
| QIAquick PCR Purification Kit (250) | QIAGEN | 28106 | |
| mMESSAGE mMACHINE SP6 Transcription Kit | Ambion | AM1340 | |
| BL21 (DE3)-T-1 competent cell | Sigma-Aldrich | B2935 | |
| Calcium ionophore | Sigma-Aldrich | A23187 | |
| Hoechst 33342 | Sigma-Aldrich | B2261 | Toxic |
| Trichloro | Sigma-Aldrich | 448931 | Toxic |
| (1H,1H,2H,2H-perfluorooctyl) silane | |||
| PFPE-PEG surfactant | Ran Biotechnologies | 008-FluoroSurfactant-2wtH-50G | |
| GE Healthcare Glutathione Sepharose 4B beads | Sigma-Aldrich | GE17-0756-01 | |
| PD-10 column | Sigma-Aldrich | GE17-0851-01 | |
| VitroCom miniature hollow glass tubing | VitroCom | 5012 | |
| Olympus SZ61 Stereo Microscope | Olympus | ||
| Olympus IX83 microscope | Olympus | ||
| Olympus FV1200 confocal microscope | Olympus | ||
| NanoDrop spectrophotometer | Thermofisher | ND-2000 | |
| 0.4 mL Snap-Cap Microtubes | E&K Scientific | 485050-B | |
| PureLink RNA Mini Kit | ThermoFisher（Ambion） | 12183018A | |
| Fisherbrand Analog Vortex Mixer | Fisher Scientific | 2215365 | |
| Imaris | Bitplane | Version 7.3 | Image analysis software |
Referencias
- Murray, A. W. Cell cycle extracts. Methods in Cell Biology. 36, 581-605 (1991).
- Hannak, E., Heald, R. Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts. Nature Protocols. 1, 2305-2314 (2006).
- Murray, A. W., Solomon, M. J., Kirschner, M. W. The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature. 339, 280-286 (1989).
- Yang, Q., Ferrell, J. E. The Cdk1-APC/C cell cycle oscillator circuit functions as a time-delayed, ultrasensitive switch. Nature Cell Biology. 15, 519-525 (2013).
- Chang, J. B., Ferrell, J. E. Mitotic trigger waves and the spatial coordination of the Xenopus cell cycle. Nature. 500, 603-607 (2013).
- Trunnell, N. B., Poon, A. C., Kim, S. Y., Ferrell, J. E. Ultrasensitivity in the Regulation of Cdc25C by Cdk1. Molecular Cell. 41, 263-274 (2011).
- Kim, S. Y., Ferrell, J. E. Substrate competition as a source of ultrasensitivity in the inactivation of Wee1. Cell. 128, 1133-1145 (2007).
- Pomerening, J. R., Kim, S. Y., Ferrell, J. E. Systems-level dissection of the cell-cycle oscillator: bypassing positive feedback produces damped oscillations. Cell. 122, 565-578 (2005).
- Pomerening, J. R., Sontag, E. D., Ferrell, J. E. Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2. Nature Cell Biology. 5, 346-351 (2003).
- Telley, I. A., Gaspar, I., Ephrussi, A., Surrey, T. Aster migration determines the length scale of nuclear separation in the Drosophila syncytial embryo. The Journal of Cell Biology. 197, 887-895 (2012).
- Telley, I. A., Gaspar, I., Ephrussi, A., Surrey, T. A single Drosophila embryo extract for the study of mitosis ex vivo. Nature Protocols. 8, 310-324 (2013).
- Tsai, T. Y. C., Theriot, J. A., Ferrell, J. E. Changes in Oscillatory Dynamics in the Cell Cycle of Early Xenopus laevis Embryos. PLoS Biology. 12, e1001788 (2014).
- Chang, J. B., Ferrell, J. E. Mitotic trigger waves and the spatial coordination of the Xenopus cell cycle. Nature. 500, 603-607 (2013).
- Yang, Q., Ferrell, J. E. The Cdk1-APC/C cell cycle oscillator circuit functions as a time-delayed, ultrasensitive switch. Nature Cell Biology. 15, 519-525 (2013).
- Kumagai, A., Dunphy, W. G. Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts. Molecular Cell. 6, 839-849 (2000).
- Chen, R. H., Murray, A. Characterization of spindle assembly checkpoint in Xenopus egg extracts. Methods in Enzymology. 283, 572-584 (1997).
- Chen, R. H., Waters, J. C., Salmon, E. D., Murray, A. W. Association of spindle assembly checkpoint component XMAD2 with unattached kinetochores. Science. 274, 242-246 (1996).
- Minshull, J., Sun, H., Tonks, N. K., Murray, A. W. A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts. Cell. 79, 475-486 (1994).
- Good, M. C., Vahey, M. D., Skandarajah, A., Fletcher, D. A., Heald, R. Cytoplasmic Volume Modulates Spindle Size During Embryogenesis. Science. 342, 856-860 (2013).
- Hazel, J., et al. Changes in cytoplasmic volume are sufficient to drive spindle scaling. Science. 342, 853-856 (2013).
- Garibyan, L., Avashia, N. Research Techniques Made Simple: Polymerase Chain Reaction (PCR). The Journal of Investigative Dermatology. 133, e6 (2013).
- Hecker, K. H., Roux, K. H. High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR. BioTechniques. 20, 478-485 (1996).
- Gibson, D. G., et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods. 6, 343-345 (2009).
- Froger, A., Hall, J. E. Transformation of Plasmid DNA into E. coli Using the Heat Shock Method. Journal of Visualized Experiments. 6, e253 (2007).
- Torreilles, S. L., McClure, D. E., Green, S. L. Evaluation and refinement of euthanasia methods for Xenopus laevis. Journal of the American Association for Laboratory Animal Science. 48, 512-516 (2009).
- Sive, H. L., Grainger, R. M., Harland, R. M. Isolating Xenopus laevis Testes. Cold Spring Harbor Protocols. 2007, (2007).
- Showell, C., Conlon, F. L. Egg Collection and In vitro Fertilization of the Western Clawed Frog Xenopus tropicalis. Cold Spring Harbor Protocols. 2009, (2009).
- Wilson, C. M. . Methods in Enzymology. 91, 236-247 (1983).
- Schutze, T., et al. A streamlined protocol for emulsion polymerase chain reaction and subsequent purification. Analytical Biochemistry. 410, 155-157 (2011).
- Weitz, M., et al. Diversity in the dynamical behaviour of a compartmentalized programmable biochemical oscillator. Nature Chemistry. 6, 295-302 (2014).
- Ho, K. K., Lee, J. W., Durand, G., Majumder, S., Liu, A. P. Protein aggregation with poly(vinyl) alcohol surfactant reduces double emulsion-encapsulated mammalian cell-free expression. PloS One. 12, e0174689 (2017).
- Nakajima, M., et al. Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science. 308, 414-415 (2005).
- Guan, Y., et al. A robust and tunable mitotic oscillator in artificial cells. eLife. 7, (2018).
Citar este artículo
Guan, Y., Wang, S., Jin, M., Xu, H., Yang, Q. Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts. J. Vis. Exp. (139), e58240, doi:10.3791/58240 (2018).