JoVE Journal > Biology
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Please note that all translations are automatically generated. Click here for the English version.
Biology

无细胞测定中使用<em>非洲爪蟾</em>胚胎提取研究核的大小调节机制。

Published: August 08, 2016
doi:
10.3791/54173
,
1Department of Molecular Biology,University of Wyoming

Summary

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

Abstract

在细胞生物学中的一个基本问题是细胞和细胞器的尺寸是如何监管。人们早已认识到，核的大小通常与该单元的大小时，在这两种细胞显着减少和核大小发生磅秤，尤其是在胚胎发生期间。核大小调控机制在很大程度上是未知的，并且可以是相关的癌症，其中改变的核的大小是关键的诊断和预后参数。 体内接近识别核大小调节由核函数的必要性和复杂性是复杂的。这里描述的研究核大小控制的体外方法考虑在核大小爪蟾发展过程中发生的正常减少的优点。首先，细胞核被组装在十蟾卵提取物。然后，这些晶核分离并再悬浮于细胞质从晚期胚胎。经过30 – 90分钟的潜伏期，核表面积20下降 – 60％，提供了有益的试验，以确定目前在后期阶段的胚胎，有助于发展核大小缩放细胞质成分。这种方法的主要优点是相对设施与该卵和胚胎提取物可生化操纵，从而允许调节核大小新型蛋白质和活动的识别。正如任何体外方法中， 在体内系统的结果的验证是重要的，和X的显微注射蟾胚胎特别适用于这些研究。

Introduction

细胞器的尺寸通常比例与电池的尺寸，这已被也许是最好的核大小的与细胞大小1-10缩放记录。这是胚胎发育和细胞分化，当这两种细胞和核大小显着减少经常观察到11,12时尤其如此。此外，改变的核尺寸是在癌症诊断和预后13-17的关键参数。有助于核大小调控机制在很大程度上是未知的，部分是由于复杂性和核的结构和功能的基本性质。这里所描述的方法被开发作为核大小缩放体外测定是适合于生物化学操作和核大小调控机制澄清。

非洲爪蟾卵提取物是一个行之有效的制度来概括和研究在体外复杂的细胞过程</em>背景。这些提取物已经透露了几个细胞过程，包括装配和有丝分裂纺锤体，内质网的功能，以及核18-22新的基础资料。向萃取系统的一个主要优点是，X。蟾卵提取物代表几乎未稀释的细胞质的组合物可以容易地改变，例如通过加入重组蛋白或免疫耗竭的。此外，一个是能够通过采用处理，否则可能会在体内上下文致死操纵基本过程。蛋提取过程的变型允许从X的提取物的隔离蟾胚胎而不是卵，这些胚胎提取物也同样适合于生物化学操作23。在X.蟾发展，单细胞受精胚胎（约1毫米直径）经历一系列十二快速细胞分裂（阶段1 – 8），以产生几千50μ米直径和较小的小区，达到一个发育阶段称为midblastula过渡（MBT）或阶段8月24日至26日。 MBT的特点是合子转录，细胞迁移，异步细胞分裂，获取间隙相的，并建立无稳态大小，而不是连续的核扩张为在预MBT胚胎的发作。从第4阶段到原肠胚形成（阶段10.5 – 12），从个人细胞核的体积超过10倍11减小。

在这里，我们的目标是，以确定负责发育进程中这些削减核大小的机制。该方法是先在组装核X.蟾卵提取物和从卵胞浆/提取那些核隔离。然后，这些核悬浮在细胞质中晚期原肠期胚胎。孵育期后，从卵提取物晶核成为在后期胚胎提取物小。我们的理由是竟被这d是用于识别存在于晚期胚胎有助于发展核大小缩放细胞质成分的有用试验。使用这种测定法，再加上在体内验证，我们表明，蛋白激酶C（PKC）有助于在十核大小发育削减蟾 23。

Protocol

所有的爪蟾程序和研究是在遵守NRC指南实验动物第8版的管理和使用进行。协议由怀俄明州机构动物护理和使用委员会（保证＃A-3216-01）的大学批准。 1. X的准备蟾卵提取物（改编自27,28） 总理的女X.蟾青蛙最少三天收蛋之前最多两个星期与一个100 IU注射孕马血清促性腺激素（PMSG）的。 实验前一天，以每1/3倍马克的修改振铃（MMR）的青蛙1升在16…

Representative Results

核的大会卵提取物 此协议的第一步是准备X.蟾卵提取物（协议1）和demembranated精子细胞核（协议2）。然后，这些试剂用于组装细胞核从头 （协议3）。 图1示出了一些有代表性的数据。钙的添加驱动减数分裂逮捕卵提取物成相间，和放线菌酮保持在间被捕的提取物。由30 – 反应开始45分钟?…

Discussion

Here is presented a novel method to study mechanisms of nuclear size regulation during X. laevis development. Developmental progression is associated with dramatic changes in cell physiology, metabolism, division rates, and migration, as well as alterations in the sizes of cells and intracellular structures. These varied processes are complex and essential, so it is difficult to study just one of these aspects of development in an in vivo setting. The X. laevis embryo extract and nuclear shrink…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Members of the Levy and Gatlin labs as well as colleagues in the Department of Molecular Biology offered helpful advice and discussions. Rebecca Heald provided support in the early stages of developing this protocol. This work was supported by the NIH/NIGMS (R15GM106318) and the American Cancer Society (RSG-15-035-01-DDC).

Materials

Alexa Fluor 568 Donkey anti-mouse IgG Molecular Probes A10037
ATP disodium salt Sigma Aldrich A2383
Benzocaine Sigma Aldrich E1501
Bovine Serum Albumin Sigma Aldrich A3059
CaCl2 Sigma Aldrich C3306
Centrifuge Beckman J2-21M
Centrifuge rotor Beckman JS 13.1
chymostatin Sigma Aldrich C7268
creatine phosphate disodium Calbiochem 2380
cycloheximide Sigma Aldrich C6255
cytochalasin D Sigma Aldrich C8273
disposable wipes (kimwipes) Sigma Aldrich Z188956
L-cysteine Sigma Aldrich W326306
EGTA Sigma Aldrich E4378
Formaldehyde Sigma Aldrich F8775
Glass crystallizing dish (150×75 mm) VWR 89090-662
Glycerol Macron 5094-16
HEPES Sigma Aldrich H4034
Hoechst – bisBenzimide H 33342 trihydrochloride Sigma Aldrich B2261
HCG – Human Chorionic Gonadotropin  Prospec hor-250-c
L15 Media Sigma Aldrich L4386
leupeptin Sigma Aldrich L2884
Lysolecithin Sigma Aldrich L1381
mAb414 Abcam ab24609
MgCl2 EMD MX0045-2
MgSO4 Sigma Aldrich M9397
Maltose Sigma Aldrich M5885
NP40 BDH 56009
Paraformaldehyde Electron Microscopy Sciences 15710
Penicillin + Streptomycin Sigma Aldrich Pp0781
pepstatin Sigma Aldrich P5318
PIPES Sigma Aldrich P6757
Plastic paraffin film (parafilm) Sigma Aldrich P7793
KCl Sigma Aldrich P9541
KH2PO4 Mallinckrodt 70100
KOH Baker 5 3140
PMSG – Pregnant Mare Serum Gonadotropin Prospec hor-272-a
NaCl Sigma Aldrich S3014
NaHCO3 Fisher BP328
NaHPO4 EMD SX0720-1
NaOH EMD SX0590
Pestle Thomas Scientific 3411D56
Round bottom glass tubes, 15 ml Corex 8441
Secondary antibody (Alexa Fluor 568 donkey anti-mouse IgG) ThermoFisher A10037
sucrose Calbiochem 8550
thermal cycler Bio-Rad T100
Ultracentrifuge Beckman L8-80M
Ultracentrifuge rotor Beckman SW 50.1
Vectashield (anti-fade mounting medium) Vector H-1000
DOWNLOAD MATERIALS LIST

References

  1. Conklin, E. Cell size and nuclear size. J. Exp. Embryol. 12, 1-98 (1912).
  2. Wilson, E. B. . The Cell in Development and Heredity. , 727-733 (1925).
  3. Levy, D. L., Heald, R. Nuclear size is regulated by importin alpha and Ntf2 in Xenopus. Cell. 143 (2), 288-298 (2010).
  4. Chan, Y. H., Marshall, W. F. Scaling properties of cell and organelle size. Organogenesis. 6 (2), 88-96 (2010).
  5. Walters, A. D., Bommakanti, A., Cohen-Fix, O. Shaping the nucleus: Factors and forces. J Cell Biochem. 113 (9), 2813-2821 (2012).
  6. Webster, M., Witkin, K. L., Cohen-Fix, O. Sizing up the nucleus: nuclear shape, size and nuclear-envelope assembly. J Cell Sci. 122 (Pt 10), 1477-1486 (2009).
  7. Edens, L. J., White, K. H., Jevtic, P., Li, X., Levy, D. L. Nuclear size regulation: from single cells to development and disease. Trends Cell Biol. 23 (4), 151-159 (2013).
  8. Jevtic, P., Edens, L. J., Vukovic, L. D., Levy, D. L. Sizing and shaping the nucleus: mechanisms and significance. Curr Opin Cell Biol. 28, 16-27 (2014).
  9. Levy, D. L., Heald, R. Mechanisms of intracellular scaling. Annu Rev Cell Dev Biol. 28, 113-135 (2012).
  10. Vukovic, L. D., Jevtic, P., Edens, L. J., Levy, D. L. New insights into the mechanisms and functions of nuclear size regulation. Int Rev Cell Mol Biol. 322, 1-59 (2016).
  11. Jevtic, P., Levy, D. L. Nuclear size scaling during Xenopus early development contributes to midblastula transition timing. Curr Biol. 25 (1), 45-52 (2015).
  12. Hara, Y., Iwabuchi, M., Ohsumi, K., Kimura, A. Intranuclear DNA density affects chromosome condensation in metazoans. Mol Biol Cell. 24 (15), 2442-2453 (2013).
  13. Zink, D., Fischer, A. H., Nickerson, J. A. Nuclear structure in cancer cells. Nat Rev Cancer. 4 (9), 677-687 (2004).
  14. Jevtic, P., Levy, D. L. Mechanisms of nuclear size regulation in model systems and cancer. Adv Exp Med Biol. 773, 537-569 (2014).
  15. Chow, K. H., Factor, R. E., Ullman, K. S. The nuclear envelope environment and its cancer connections. Nat Rev Cancer. 12 (3), 196-209 (2012).
  16. Blom, J. H., Ten Kate, F. J., Schroeder, F. H., van der Heul, R. O. Morphometrically estimated variation in nuclear size. A useful tool in grading prostatic cancer. Urol Res. 18 (2), 93-99 (1990).
  17. Dey, P. Cancer nucleus: morphology and beyond. Diagn Cytopathol. 38 (5), 382-390 (2010).
  18. Cross, M. K., Powers, M. A. Learning about cancer from frogs: analysis of mitotic spindles in Xenopus egg extracts. Dis Model Mech. 2 (11-12), 541-547 (2009).
  19. Voeltz, G. K., Prinz, W. A., Shibata, Y., Rist, J. M., Rapoport, T. A. A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell. 124 (3), 573-586 (2006).
  20. Chan, R. C., Forbes, D. I. In vitro study of nuclear assembly and nuclear import using Xenopus egg extracts. Methods Mol Biol. 322, 289-300 (2006).
  21. Edens, L. J., Levy, D. L., Kloc, M., Kubiak, J. Z. . Xenopus Development. , 325-345 (2014).
  22. Levy, D. L., Heald, R. Biological Scaling Problems and Solutions in Amphibians. Cold Spring Harb Perspect Biol. 8 (1), a019166 (2016).
  23. Edens, L. J., Levy, D. L. cPKC regulates interphase nuclear size during Xenopus development. J Cell Biol. 206 (4), 473-483 (2014).
  24. Nieuwkoop, P. D., Faber, J. . Normal Table of Xenopus laevis (Daudin). , (1967).
  25. Newport, J., Kirschner, M. A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription. Cell. 30 (3), 687-696 (1982).
  26. Newport, J., Kirschner, M. A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage. Cell. 30 (3), 675-686 (1982).
  27. Maresca, T. J., Heald, R. Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts. Methods Mol Biol. 322, 459-474 (2006).
  28. Hannak, E., Heald, R. Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts. Nat Protoc. 1 (5), 2305-2314 (2006).
  29. Murray, A. W. Cell cycle extracts. Methods Cell Biol. 36, 581-605 (1991).
  30. Khan, M. A., et al. Quantitative alterations in nuclear structure predict prostate carcinoma distant metastasis and death in men with biochemical recurrence after radical prostatectomy. Cancer. 98 (12), 2583-2591 (2003).
  31. Tan, P. H., Goh, B. B., Chiang, G., Bay, B. H. Correlation of nuclear morphometry with pathologic parameters in ductal carcinoma in situ of the breast. Mod Pathol. 14 (10), 937-941 (2001).
  32. Zeimet, A. G., et al. DNA ploidy, nuclear size, proliferation index and DNA-hypomethylation in ovarian cancer. Gynecol Oncol. 121 (1), 24-31 (2011).
  33. Theerthagiri, G., Eisenhardt, N., Schwarz, H., Antonin, W. The nucleoporin Nup188 controls passage of membrane proteins across the nuclear pore complex. J Cell Biol. 189 (7), 1129-1142 (2010).
  34. Wuhr, M., et al. Evidence for an upper limit to mitotic spindle length. Curr Biol. 18 (16), 1256-1261 (2008).
  35. Loughlin, R., Wilbur, J. D., McNally, F. J., Nedelec, F. J., Heald, R. Katanin contributes to interspecies spindle length scaling in Xenopus. Cell. 147 (6), 1397-1407 (2011).
  36. Wilbur, J. D., Heald, R. Mitotic spindle scaling during Xenopus development by kif2a and importin. eLife. 2, e00290 (2013).
  37. Kieserman, E. K., Heald, R. Mitotic chromosome size scaling in Xenopus. Cell Cycle. 10 (22), 3863-3870 (2011).
  38. Maresca, T. J., Freedman, B. S., Heald, R. Histone H1 is essential for mitotic chromosome architecture and segregation in Xenopus laevis egg extracts. J Cell Biol. 169 (6), 859-869 (2005).
  39. MacCallum, D. E., Losada, A., Kobayashi, R., Hirano, T. ISWI remodeling complexes in Xenopus egg extracts: identification as major chromosomal components that are regulated by INCENP-aurora B. Mol Biol Cell. 13 (1), 25-39 (2002).
  40. Goehring, N. W., Hyman, A. A. Organelle growth control through limiting pools of cytoplasmic components. Curr Biol. 22 (9), R330-R339 (2012).
  41. Du Toit, A. Development: Honey, I shrunk the nucleus. Nat Rev Mol Cell Biol. 15 (10), 633 (2014).
  42. Buchner, K. The role of protein kinase C in the regulation of cell growth and in signalling to the cell nucleus. J Cancer Res Clin Oncol. 126 (1), 1-11 (2000).
  43. Mochly-Rosen, D., Das, K., Grimes, K. V. Protein kinase C, an elusive therapeutic target. Nat Rev Drug Discov. 11 (12), 937-957 (2012).

Tags

Xenopus LaevisCell-free AssayNuclear Size RegulationEmbryo ExtractsSperm DemembranationEgg FertilizationDejellyingEmbryo Staging

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Cite This Article
Edens, L. J., Levy, D. L. A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation. J. Vis. Exp. (114), e54173, doi:10.3791/54173 (2016).
Download Citation
Reprints and Permissions

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image