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Biologia

非洲爪蟾 用于可视化动态细胞质组织的蛋提取物制备和实时成像方法

Published: June 06, 2021
doi:
10.3791/61923
,
1Department of Chemical and Systems Biology,Stanford University School of Medicine, 2Current Address: Department of Biological Sciences,University of Southern California, 3Department of Biochemistry,Stanford University School of Medicine

Summary

我们描述了一种从 非洲爪蟾 卵中制备和实时成像未稀释的细胞质提取物的方法。

Abstract

非洲 爪蟾 卵提取物传统上用于批量生化测定，已成为一种强大的基于成像的工具，用于研究细胞质现象，例如细胞分裂、有丝分裂纺锤体形成和细胞核组装。基于对在稀疏时间点采样的固定提取物进行成像的早期方法，最近的方法使用延时显微镜对实时提取物进行成像，揭示了具有增强时间分辨率的更多动态特征。这些方法通常需要对成像容器进行复杂的表面处理。在这里，我们介绍了一种无需化学表面处理的鸡蛋提取物实时成像的替代方法。它易于实施，并利用批量生产的实验室耗材进行成像。我们描述了一种可用于宽场和共聚焦显微镜的系统。它专为二维（2D）场中的提取物成像而设计，但可以轻松扩展到3D成像。它非常适合研究细胞质内的空间模式形成。通过代表性数据，我们证明了使用该方法制备的相间提取物中微管，细胞核和线粒体的典型动态组织。这些图像数据可以提供有关细胞质动力学和空间组织的定量信息。

Introduction

细胞质构成细胞的主要体积，具有独特的组织。真核细胞质的成分可以自组装成广泛的空间结构，例如微管紫苑和高尔基体，而微管紫苑又根据细胞的身份和生理状态动态排列和翻转。因此，了解细胞质的空间组织及其与细胞功能的联系对于理解细胞如何工作非常重要。非洲爪蟾卵提取物传统上用于批量生化测定1，2，3，4，5，6，7，8，但最近的工作将它们确立为强大的实时成像系统，用于细胞质结构及其细胞功能的机制研究9，10，11，12，13，14，15，16，17，18.这些未稀释的提取物保留了细胞质的许多结构和功能，同时允许直接操作细胞质内容物，这是传统基于细胞的模型无法实现的19，20。这使得它们成为表征细胞质现象和剖析其机制基础的理想选择。

现有的提取物成像方法需要化学表面改性或制造微流体装置。一种基于盖玻片的方法需要对玻璃盖玻片进行聚乙二醇（PEG）钝化21。基于微乳液的方法需要在玻璃表面上气相沉积三氯（1H，1H，2H，2H-全氟辛基）硅烷22，23。基于微流体的系统可以精确控制提取物液滴的体积、几何形状和组成，但需要专门的微细加工设施11，12，24。

在这里，我们介绍了一种对卵子提取物进行成像的替代方法，该方法易于实施并利用现成的低成本材料。这包括制备带有载玻片和涂有氟化乙烯丙烯（FEP）胶带的盖玻片的成像室。该腔室可用于使用各种显微镜系统对提取物进行成像，包括立体镜以及正置和倒置显微镜。该方法不需要对表面进行化学处理，同时获得与上述现有玻璃方法相似的光学透明度。它旨在对 2D 视场中厚度均匀的提取物层进行成像，并且可以轻松扩展以对提取物的 3D 体积进行成像。它非常适合在大视场上对集体细胞质行为进行延时成像。

我们使用相间停滞的卵提取物来演示我们的成像方法。提取物制备遵循戴明和科恩布鲁斯19的方案。简而言之，在减数分裂II中期自然停滞的卵子被低速旋转压碎。该旋转将细胞质从减数分裂停滞中释放出来，并允许提取物进入间期。通常，在粉碎自旋之前添加细胞松弛素B以抑制F-肌动蛋白的形成。然而， 如果需要 F-肌动蛋白， 它可以省略.在粉碎旋转之前还添加环己酰亚胺，以防止中间提取物进入下一个有丝分裂。随后将提取物置于上述成像室中并置于显微镜上。最后，通过连接到显微镜的相机以定义的时间间隔记录图像，生成延时图像系列，在2D场中捕获提取物的动态行为。

Protocol

此处描述的所有方法均已获得斯坦福大学机构动物护理和使用委员会（IACUC）的批准。 1. 载玻片和盖玻片的准备 用滚筒涂抹器将一层氟化乙烯丙烯（FEP）胶带涂在载玻片上。用干净的剃须刀片剪掉边缘上多余的胶带。以相同的方式制备FEP胶带包被的盖玻片（图1A）。 将双面粘性成像垫片涂在载玻片的FEP胶带涂层面上。保持顶部的保护衬垫…

Representative Results

非洲爪蟾卵提取物可用于研究间期细胞质的自组织。图2A显示了成功实验的结果。我们用浓度为 27 个细胞核/μL 的去膜爪蟾精子核19 和 0.38 μM 纯化的 GST-GFP-NLS27、28、29、30（由谷胱甘肽-S-转移酶、绿色荧光蛋白和核定位序列组成的?…

Discussion

非洲爪蟾卵提取物已成为一种强大的模型系统，用于各种亚细胞结构的成像研究10，14，15，16，17，18，21，31，32，33，34，35<su…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

我们感谢J. Kamenz、Y. Chen和W. Y. C. Huang对手稿的评论。这项工作得到了美国国立卫生研究院（R01 GM110564，P50 GM107615和R35 GM131792）的资助，授予James E. Ferrell，Jr.。

Materials

17 ml centrifuge tube Beckman Coulter 337986
22×22 mm square #1 cover glass Corning 284522
Aprotinin MilliporeSigma 10236624001 Protease inhibitor
Cycloheximide MilliporeSigma 01810 Protein synthesis inhibitor
Cytochalasin B MilliporeSigma C6762 Actin polymerization inhibitor
Female Xenopus laevis frogs Nasco LM00535MX
Fluorescent HiLyte 488 labeled tubulin protein Cytoskeleton, Inc. TL488M-A For visualizing the microtubule cytoskeleton
Fluorescent HiLyte 647 labeled tubulin protein Cytoskeleton, Inc. TL670M-A For visualizing the microtubule cytoskeleton
Fluorinated ethylene propylene (FEP) optically clear tape CS Hyde company 23-FEP-2-5
Glass Pasteur pipette Fisher Scientific 13-678-20C
Human chorionic gonadotropin (hCG) MilliporeSigma CG10
Imaging spacer Electron Microscopy Sciences 70327-8S
Leupeptin MilliporeSigma 11017101001 Protease inhibitor
Microscope slides Fisher Scientific 12-518-100B
Mineral oil MilliporeSigma 330760
MitoTracker Red CMXRos Thermo Fisher Scientific M7512 For visualizing mitochondria
Pregnant mare serum gonadotropin (PMSG) BioVendor RP1782725000
Roller applicator Amazon B07HMBJSP8 For applying the FEP tape to the glass slides and coverslips
Single-edged razor blades Fisher Scientific 12-640 For removing excessive FEP tape
Transfer pipette Fisher Scientific 13-711-7M
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Riferimenti

  1. Murray, A. W., Kirschner, M. W. Cyclin synthesis drives the early embryonic cell cycle. Nature. 339 (6222), 275-280 (1989).
  2. Dunphy, W. G., Brizuela, L., Beach, D., Newport, J. The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell. 54 (3), 423-431 (1988).
  3. Minshull, J., Golsteyn, R., Hill, C. S., Hunt, T. The A- and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle. EMBO J. 9 (9), 2865-2875 (1990).
  4. Wu, J. Q., et al. PP1-mediated dephosphorylation of phosphoproteins at mitotic exit is controlled by inhibitor-1 and PP1 phosphorylation. Nature Cell Biology. 11 (5), 644-651 (2009).
  5. 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 (4), 346-351 (2003).
  6. Mochida, S., Maslen, S. L., Skehel, M., Hunt, T. Greatwall phosphorylates an inhibitor of protein phosphatase 2A that is essential for mitosis. Science. 330 (6011), 1670-1673 (2010).
  7. Blow, J. J., Laskey, R. A. Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell. 47 (4), 577-587 (1986).
  8. Lohka, M. J., Masui, Y. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science. 220 (4598), 719-721 (1983).
  9. Cheng, X., Ferrell, J. E. Spontaneous emergence of cell-like organization in Xenopus egg extracts. Science. 366 (6465), 631-637 (2019).
  10. Nguyen, P. A., et al. Spatial organization of cytokinesis signaling reconstituted in a cell-free system. Science. 346 (6206), 244-247 (2014).
  11. Good, M. C., Vahey, M. D., Skandarajah, A., Fletcher, D. A., Heald, R. Cytoplasmic volume modulates spindle size during embryogenesis. Science. 342 (6160), 856-860 (2013).
  12. Hazel, J., et al. Changes in cytoplasmic volume are sufficient to drive spindle scaling. Science. 342 (6160), 853-856 (2013).
  13. Brownlee, C., Heald, R. Importin alpha Partitioning to the Plasma Membrane Regulates Intracellular Scaling. Cell. 176 (4), 805-815 (2019).
  14. Nguyen, P. A., Field, C. M., Mitchison, T. J. Prc1E and Kif4A control microtubule organization within and between large Xenopus egg asters. Molecular Biology of the Cell. 29 (3), 304-316 (2018).
  15. Desai, A., Maddox, P. S., Mitchison, T. J., Salmon, E. D. Anaphase A chromosome movement and poleward spindle microtubule flux occur At similar rates in Xenopus extract spindles. Journal of Cell Biology. 141 (3), 703-713 (1998).
  16. Mitchison, T. J., et al. Roles of polymerization dynamics, opposed motors, and a tensile element in governing the length of Xenopus extract meiotic spindles. Molecular Biology of the Cell. 16 (6), 3064-3076 (2005).
  17. Mitchison, T. J., et al. Bipolarization and poleward flux correlate during Xenopus extract spindle assembly. Molecular Biology of the Cell. 15 (12), 5603-5615 (2004).
  18. Murray, A. W., Desai, A. B., Salmon, E. D. Real time observation of anaphase in vitro. Proceedings of the National Academy of Sciences of the United States of America. 93 (22), 12327-12332 (1996).
  19. Deming, P., Kornbluth, S. Study of apoptosis in vitro using the Xenopus egg extract reconstitution system. Methods in Molecular Biology. 322, 379-393 (2006).
  20. Murray, A. W. Cell cycle extracts. Methods in Cell Biology. 36, 581-605 (1991).
  21. Field, C. M., Mitchison, T. J. Assembly of Spindles and Asters in Xenopus Egg Extracts. Cold Spring Harbor Protocols. 2018 (6), (2018).
  22. Guan, Y., et al. A robust and tunable mitotic oscillator in artificial cells. Elife. 7, (2018).
  23. Guan, Y., Wang, S., Jin, M., Xu, H., Yang, Q. Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts. Journal of Visualized Experiments. (139), e58240 (2018).
  24. Oakey, J., Gatlin, J. C. Microfluidic Encapsulation of Demembranated Sperm Nuclei in Xenopus Egg Extracts. Cold Spring Harbor Protocols. 2018 (8), (2018).
  25. Smythe, C., Newport, J. W. Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts. Methods in Cell Biology. 35, 449-468 (1991).
  26. Field, C. M., et al. Actin behavior in bulk cytoplasm is cell cycle regulated in early vertebrate embryos. Journal of Cell Science. 124, 2086-2095 (2011).
  27. Chang, J. B., Ferrell, J. E. Mitotic trigger waves and the spatial coordination of the Xenopus cell cycle. Nature. 500 (7464), 603-607 (2013).
  28. Chang, J. B., Ferrell, J. E. Robustly Cycling Xenopus laevis Cell-Free Extracts in Teflon Chambers. Cold Spring Harbor Protocols. 2018 (8), (2018).
  29. Chatterjee, S., Javier, M., Stochaj, U. In vivo analysis of nuclear protein traffic in mammalian cells. Experimental Cell Research. 236 (1), 346-350 (1997).
  30. Mochida, S., Hunt, T. Calcineurin is required to release Xenopus egg extracts from meiotic M phase. Nature. 449 (7160), 336-340 (2007).
  31. Heald, R., et al. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature. 382 (6590), 420-425 (1996).
  32. Helmke, K. J., Heald, R. TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts. Journal of Cell Biology. 206 (3), 385-393 (2014).
  33. Sawin, K. E., Mitchison, T. J. Mitotic spindle assembly by two different pathways in vitro. Journal of Cell Biology. 112 (5), 925-940 (1991).
  34. Belmont, L. D., Hyman, A. A., Sawin, K. E., Mitchison, T. J. Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell. 62 (3), 579-589 (1990).
  35. Krauss, S. W., Lee, G., Chasis, J. A., Mohandas, N., Heald, R. Two protein 4.1 domains essential for mitotic spindle and aster microtubule dynamics and organization in vitro. Journal of Biological Chemistry. 279 (26), 27591-27598 (2004).
  36. Wang, S., Romano, F. B., Field, C. M., Mitchison, T. J., Rapoport, T. A. Multiple mechanisms determine ER network morphology during the cell cycle in Xenopus egg extracts. Journal of Cell Biology. 203 (5), 801-814 (2013).
  37. Field, C. M., Nguyen, P. A., Ishihara, K., Groen, A. C., Mitchison, T. J. Xenopus egg cytoplasm with intact actin. Methods in Enzymology. 540, 399-415 (2014).
  38. Good, M. C., Heald, R. Preparation of Cellular Extracts from Xenopus Eggs and Embryos. Cold Spring Harbor Protocols. 2018 (6), (2018).
  39. Field, C. M., Pelletier, J. F., Mitchison, T. J. Xenopus extract approaches to studying microtubule organization and signaling in cytokinesis. Methods in Cell Biology. 137, 395-435 (2017).

Tags

Xenopus LaevisEgg ExtractLive ImagingCytoplasmic OrganizationFEP TapeImaging SpacerDejellying SolutionMMR BufferEgg Lysis BufferCentrifugationAprotininLeupeptinCytochalasin BCyclohexamideCytoplasmic Layer
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Citazione di questo articolo
Cheng, X., Ferrell, Jr., J. E. Xenopus laevis Egg Extract Preparation and Live Imaging Methods for Visualizing Dynamic Cytoplasmic Organization. J. Vis. Exp. (172), e61923, doi:10.3791/61923 (2021).
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