Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
Nat Protoc. 2013 Feb;8(2):310-24. doi: 10.1038/nprot.2013.003. Epub 2013 Jan 17.
Spindle assembly and chromosome segregation rely on a complex interplay of biochemical and mechanical processes. Analysis of this interplay requires precise manipulation of endogenous cellular components and high-resolution visualization. Here we provide a protocol for generating an extract from individual Drosophila syncytial embryos that supports repeated mitotic nuclear divisions with native characteristics. In contrast to the large-scale, metaphase-arrested Xenopus egg extract system, this assay enables the serial generation of extracts from single embryos of a genetically tractable organism, and each extract contains dozens of autonomously dividing nuclei that can be prepared and imaged within 60-90 min after embryo collection. We describe the microscopy setup and micropipette production that facilitate single-embryo manipulation, the preparation of embryos and the steps for making functional extracts that allow time-lapse microscopy of mitotic divisions ex vivo. The assay enables a unique combination of genetic, biochemical, optical and mechanical manipulations of the mitotic machinery.
纺锤体组装和染色体分离依赖于生化和机械过程的复杂相互作用。这种相互作用的分析需要对内源细胞成分进行精确的操作和高分辨率的可视化。在这里，我们提供了一种从单个果蝇合胞胚胎中产生提取物的方案，该提取物支持具有天然特征的重复有丝分裂核分裂。与大规模、中期被阻止的爪蟾卵提取物系统相比，该测定法能够从遗传上可操作的单个胚胎中连续产生提取物，并且每个提取物包含数十个自主分裂的核，这些核可以在胚胎收集后 60-90 分钟内进行制备和成像。我们描述了显微镜设置和微吸管的生产，这些设置和生产有助于进行单个胚胎的操作，制备胚胎以及制作功能提取物的步骤，这些提取物允许对体外有丝分裂分裂进行延时显微镜观察。该测定法能够对有丝分裂机制进行独特的遗传、生化、光学和机械操作组合。
