观察有丝分裂和动力学在现场斑马鱼胚胎
Summary
Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.
Abstract
有丝分裂为有机体的生长和分化至关重要。这个过程是高度动态的，需要有序的事件来完成适当的染色质凝聚，微管连接着丝粒，染色体分离和胞质分裂在小时间框架。在微妙的过程中的错误可能导致人类疾病，包括出生缺陷和癌症。传统的方法研究人类疾病的有丝分裂状态往往依赖于细胞培养系统，研究人类疾病时，缺乏自然的生理和发育/组织特异性环境有利。该协议提供了一种可视化，高分辨率，在脊椎动物系统染色体动态，斑马鱼克服许多障碍。该协议将详细可用于获得分裂的细胞，其包括的动态图像的方法： 在体外转录，斑马鱼养殖/收集，胚胎嵌入，和延时成像。优化和改性的该协议的ications进行了探讨。使用H2A.F / Z-EGFP（标签染色质）和mCherry-CAAX（标签细胞膜）的mRNA注射的胚胎，有丝分裂在AB野生型，auroraB hi1045 和 esco2 hi2865突变斑马鱼被可视化。在斑马鱼高分辨率实时成像允许一个观察多个核分裂进行统计量化有丝分裂的缺陷和有丝分裂进程的时机。此外，定义不当的有丝分裂过程（ 即，板集合缺陷，染色体missegregation 等 ）和不适当的染色体的结果（ 即，非整倍体，多倍体，微核等 ）质量方面观察被观察。该测定可用于组织分化/发展的观察，是适合于使用突变斑马鱼和药理学试剂的。在有丝分裂的缺陷如何导致癌症和发育障碍将极大地可视化提高疾病的发病机理的理解。
Introduction
有丝分裂为生长，分化和再生在活生物体中的关键的细胞过程是必不可少的。在准确的准备和在间DNA的复制，使细胞被起动来划分。有丝分裂，前期，第一阶段是由细胞周期蛋白B / CDK1的活化启动。前期的特征在于染色材料的缩合到染色体。核膜破裂发生在前期和前中期之间的过渡。在早中期，中心体，主轴形成成核中心，开始迁移到对立的两极，而在寻找着丝点附件延伸的微管。一旦附件转换到最终的微管附着和张力定位形成中期板1染色体。如果所有染色体正确连接时，纺锤体装配检验点满足，黏着环抱着姐妹染色单体一起被切开，微管缩短拉妹妹染色单体到对立的两极后期2,3时。最后阶段，末期，涉及围绕两个新的核核膜的细胞和改革的延伸。胞质分裂完成通过分离两个新的子细胞4-6的细胞质分裂过程。关键有丝分裂途径（ 即纺锤体装配检验点，中心体复制，姐妹染色单体凝聚力等 ）的改变可能会导致中期逮捕，染色体missegregation和基因组不稳定性7-10。最终，在通路控制有丝分裂的缺陷会导致发育障碍和癌症，有丝分裂的迫使可视化和其在现场，脊椎动物，多细胞生物体10-16缺陷。
斑马鱼的胚胎作为实时成像一个伟大的模式生物，由于透明组织，易于注射的，快速发展。使用斑马鱼，这份手稿的总体目标是描述的活5D有丝分裂17（ 图1C）的（尺寸的X，Y，Z，时间和波长）成像的方法。利用斑马鱼突变体在不同有丝分裂的途径有缺陷的论证这种缺陷的结果。对于这个协议，极光B和Esco2突变体被选择来说明这些缺陷。极光B是一种激酶是参与纺锤体形成和微管附着的染色体乘客复合物（CPC）的一部分。它也需要在胞质18,19分裂沟的形成。在斑马鱼中，极光B缺乏导致皱纹的诱导，细胞分裂和染色体分离20的缺陷。 Esco2，另一方面，是一种乙酰这对姐妹染色单体凝聚力21,22是必不可少的。它乙酰化上从而稳定黏结，以确保正确的染色体分离的中期-后期过渡23环的SMC3部黏着。在斑马鱼Esco2的丧失导致为chromosome missegregation，过早姐妹染色单体分离，基因组不稳定和p53依赖性和独立的凋亡24,25。由于可用性，auroraB hi1045 和 esco2 hi2865突变斑马鱼（以下简称为aurB M / M和esco2 米/分 ，分别地）将被用来说明此技术25-27。
用荧光标记的细胞的机械耦合共焦显微镜使研究人员有丝分裂25,28,29中可视化染色和细胞膜动力学。荧光标记的组蛋白在历史上被用于可视化的染色质。组蛋白是四种不同的对（H2A，H2B，H3和H4）是负责该构成染色体30中的核小体结构组成的核蛋白质。尽管H2B可以说是在荧光蛋白中最常用的组蛋白小鼠和细胞培养，用组蛋白2A的，家庭Z（H2A.F / Z）已在斑马鱼31,32使用证明很好。伴刀豆球蛋白A和酪蛋白激酶1-γ，例如，定位于细胞膜和先前已显示有效的可视化细胞膜在海胆和果蝇33,34。其它研究已经表明，CAAX荧光标记蛋白标记的细胞膜并成功地斑马鱼31。 CAAX是受翻译后修饰的酶如farnesyltransferases和geranylgeranyltransferases识别的基序。由这些酶修饰导致蛋白质成为膜相关，从而标记的细胞膜35。
由于在斑马鱼的先前使用，该协议选择使用H2A.F / Z和CAAX标记染色质和细胞膜。这种方法的应用将允许研究者在单个细胞水平监测的有丝分裂，观察个人染色体动力学，以及同时监测可能影响组织的分化和发育的多个细胞分裂。本文将着眼于在单个单元格级别的有丝分裂过程中染色体成像分离的动态。在这个手稿，观察几个有丝分裂，分裂计算时间和破译的有丝分裂表型的能力进行说明和讨论。通过使用这些参数，生理学相关的数据可以被收集并施加到受有丝分裂的缺陷数的疾病状态。
Protocol
Representative Results
Discussion
该方法的使用允许人们推断核膜破裂，通过微管的着丝点的附件形成中期板的，和姐妹染色单体的分离，以形成在体内和一个时间依赖性两个新的细胞。在斑马鱼中观察到的有丝分裂的能力是在固定的样品和细胞培养系统有利的，因为细胞在自然生理被成像，组织是透明其允许要使用的荧光蛋白质，他们开发比较快，和延时成像可以获取。成年斑马鱼的这个协议的使用是由于可能因被收购?…
Declarações
The authors have nothing to disclose.
Acknowledgements
We thank Kristen Kwan for the pCS2-H2A.F/Z-EGFP and pCS2-mCherry-CAAX vectors. We thank Chris Rodesch for tutoring us in live imaging in zebrafish. We thank Shawn Williams, Erik Malarkey and Brad Yoder for assistance in confocal imaging at UAB and the High Resolution Imaging Facility at UAB. The High Resolution Imaging Facility is supported by the UAB Comprehensive Cancer Center Support Grant (P30CA013148) and the Rheumatic Disease Core Center (P30 AR048311). J.M.P. is supported by the National Institute of Neurological Disease and Stroke (NIH R21 NS092105), and pilot grants from American Cancer Society (ACS IRG-60-001-53-IRG) and the UAB Comprehensive Cancer Center (P30CA013148). S.M.P. is supported by the Cell and Molecular Biology T32 Training Grant (5T32GM008111-28).
Materials
| pCS2 vectors | Gift from K. Kwan | For plasmid of interest | |
| NotI-HF restriction enzyme | New England BioLabs | R3189S | For restriction digest of plasmid |
| mMessage SP6 kit | Life Technologies | AM1340 | For in vitro transcription |
| RNeasy Mini kit | Qiagen | 74104 | For purifying mRNA |
| 100 x 15 mm petri dishes | Fisher Scientific | FB0875712 | For housing embryos |
| microinjection mold | homemade | For holding embryos during microinjection | |
| Agarose II | Amresco | 0815-25G | For embedding embryos |
| Tricaine | Sigma-Aldrich | E10521-10G | For anesthetizing embryos |
| Sodium Chloride | Sigma-Aldrich | S9888 | For embryo water (E3 Blue), dissolved in UltraPure H2O |
| Potassium Chloride | Sigma-Aldrich | P3911 | For embryo water (E3 Blue), dissolved in UltraPure H2O |
| Calcium Chloride Dihydrate | Sigma-Aldrich | C8106 | For embryo water (E3 Blue), dissolved in UltraPure H2O |
| Magnesium Sulfate | Fisher Scientific | M7506 | For embryo water (E3 Blue), dissolved in UltraPure H2O |
| Methylene Blue Hydrate | Sigma-Aldrich | MB1 | For embryo water (E3 Blue), dissolved in UltraPure H2O |
| 100 mm culture tube | Fisher Scientific | 50-819-812 | For melted agar |
| 35 mm glass coverslip bottom culture dish | MatTek Corp | P35G-0-20-C | No. 0, 20 mm glass, For embedding embryos |
| #5 tweezers | Dumont | 72701-D | For dechorionating embryos |
| 21G 1 1/2 gauge needle | Becton Dickinson | 305167 | For positioning embryos in agar |
| Dissecting microscope | Nikon AZ100 | For screening and embedding embryos, any dissecting scope will do | |
| Confocal microscope | Nikon A1+ | For time-lapse imaging | |
| Confocal software | NIS Elements AR 4.13.00 | For image acquisition and processing |
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